[5] Raman and resonance raman spectroscopy

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

[5] R a m a n

and Resonance

Raman

67

Spectroscopy

By HAROLD E. VAN WART and HAROLD A. SCHERAGA Introduction in the brief time that has elapsed since Raman spectroscopy (RS)' was last reviewed in this series,-' it has blossomed into a technique capable of providing detailed knowledge about the structure and conformation of enzymes. The emergence during the last few years o f r e s o n a n c e Raman spectroscopy (rRS) as a biophysical tool has been even more rapid. This recent increase in the use of RS and rRS for the study of biologically important molecules has been due in large part to the availability of commercial laser-equipped R spectrometers. In fact, high-quality R spectra can now be obtained as easily as infrared spectra. As a result, RS and rRS can be added to the list of readily accessible techniques (such as infrared and ultraviolet-visible spectroscopy, nuclear magnetic and electron spin resonance spectroscopy) that the biophysicist can use to probe the structure and conformation of biomolecules. This chapter will provide a general description of the procedures and considerations involved in carrying out and interpreting R and rR experiments on biological molecules. First, we shall review briefly the basic theoretical and experimental features of these techniques. Then, we shall discuss the interpretation of the spectra with the use of illustrative exampies. The reader should refer to other recent reviews, :~~ and references cited therein, for topics and details not dealt with here.

General Description of R a m a n and Resonance Raman Scattering and Their Relationship to Other Spectroscopic Processes When a monochromatic beam of photons of frequency ~'o impinges on a sample of matter, the electric and magnetic fields of the radiation can interact in a number of ways with the molecules in the sample. These interactions may result in the absorption of the incident photons or proAbbreviations to be used; R, Raman: S, spectro~opy: r, resonance. M. C. Tobin, this series Vol. 26 [23]. :~B. G. Frushour and J. L. Koenig, in "Advances in Infrared and Raman Spectroscopy" IR. J. H. Clark and R. E. Hester, eds.)~ Vol. I, p. 35. Heyden. London, 1975. 4T. G. Spiro and T. M. Loehr, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, eds.), Vol. 1, p. 98. Heyden, London, 1975.

68

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

duce other photons that exit from the sample with a distribution of frequencies, polarizations, intensities, and directions. The analysis of the incident and exiting photons forms the basis for all spectroscopic techniques and yields information about the eigenstates of the molecules. These eigenstates can pertain to the nuclear positions, the electronic positions, or the nuclear or electronic spins of the molecules. Ordinary, one-photon spectroscopy involves the study of molecules that absorb (or emit) photons whose energies (E = hv) exactly match the energy difference between two of their eigenstates. These are direct single-photon absorptions (or emissions). Fortunately, the energies typically associated with transitions between different rotational, vibrational, electronic or (when in the presence of an applied external magnetic field) spin states are sufficiently distinct from each other that each can be studied almost separately ~ by using photons of the appropriate frequency. For example, when single-photon absorption of radiation in the microwave, infrared, or visible-ultraviolet regions of the spectrum occurs, molecules are promoted to excited rotational, vibrational, or electronic states, respectively. Alternatively, transitions between various eigenstates can be achieved by two-photon processes that involve the simultaneous interaction of two photons with the molecule. Possible two-photon processes include the simultaneous absorption of two photons, the simultaneous emission of two photons, or the simultaneous absorption of one photon and emission of one. At normal light intensities, such two-photon processes are inherently less probable than those involving a single photon. In a two-photon process, the energy of neither of the two participating photons is equal to that of the transition. Instead, the transition energy is equal to the sum or difference of the energies of the two photons. In two-photon absorption (emission), a pair of photons whose combined energies (E = hvl + hv2) correspond to that of the transition are absorbed (emitted). R scattering is a two-photon process involving simultaneously a one-photon absorption (hVl) and a one-photon emission (hv2) in which the difference in the energies of the two photons (E = hvl - hv2) corresponds to the energy of the transition. It should be pointed out that, while two-photon transitions are achieved conceptually in two steps, the two events are experimentally inseparable in time, and it does not make sense to speak of either by itself. RS is applicable to the study of rotational, vibrational, and electronic (as well as other types of) transitions, but the discussion here will be We say "almost separately" because pure electronic or pure vibrational transitions are uncommon. In general, electronic transitions are accompanied by changes in the vibrational and rotational states also (i.e., "rovibronic'" transitions). Similarly, vibrational excitation is usually accompanied by rotational excitation.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

69

limited to the vibrational R effect in which Av 1:1 12 2 is in roughly the 10-3000 cm ~ range. The information thus obtained is complementary to that of infrared spectroscopy in which the direct, one-photon absorption of quanta with frequencies in the 10-3000 cm ~ range is studied. In Stokes R scattering, energy is transferred from the radiation field to the molecule, resulting in molecular excitation and the production of photons of lower frequency (v~ > vz), whereas in anti-Stokes R scattering energy is transferred from the molecule to the radiation field (v., > vl). Rayleigh scattering is a two-photon process, similar to R scattering in which v~ = u._,and, hence, in which there is no net energy exchange between the radiation field and the sample. To distinguish between a two-photon process and a sequence of two one-photon processes, it is necessary to consider the time dependence of the photon-molecule interaction. In fluorescence, for example, molecules are first raised to excited electronic states by absorption of photons. Then, after a measurable time delay (10 -'~' to 10-~ sec) due to the lifetime of an experimentally observable excited electronic state, the photons are reemitted, Fluorescence, then, is considered to be a sequence o f two independent one-photon processes. In R scattering, on the other hand, there is no measurable time delay between the absorption of the first photon and the emission of the second, and the molecule cannot actually be found to exist in the excited state. Hence, R scattering is a two-photon process. The various one- and two-photon processes discussed here are illustrated diagrammatically in Fig. 1. It is important to note that scattering processes proceed through "'int e r m e d i a t e " excited electronic states, That is, the absorption of the first photon brings the molecule into any of a whole set of excited electronic states (one of the many possible excited states being shown in Fig. 1), from which it returns by emission of the second photon. These intermediate states are real electronic eigenstates of the molecule (i.e., the wavefunctions describing them are solutions to the Schr6dinger equation). The nature of the involvement of intermediate states in nonresonance and resonance scattering (between which we have so far not distinguished) is quite different and can be used to provide an understanding of the differences between these two processes. In nonresonance scattering, the energy of the incident photons (E,I) is not equal to that of an allowed electronic transition. In other words, the wavelength of the incident light does not fall under an electronic absorption band of the molecule. It therefore seems to contradict the principle of conservation of energy to speak of the absorption of a photon of energy E~ (from the photon beam) causing a transition of energy Er (the energy gap from the ground to the rth excited electronic state), where Eo # E,.. How=

70

CONFORMATION:

OPTICAL SPECTROSCOPY

A t ~ 10-7-- I0 - I 0

[5]

sec

H~Ii//HI/i//N//MHMt,~N

~

~

/

1

.

/

r

/

A t <,O-12se c

D,,~= ~ -

Er i

'

~,,

;~,o

il

~

'

II

t',.,,2:

I

h,o

hl, I

,

v//////,

Em

"///ll~//ii]ii//i//il

(o)

VllJ i / i ,

(b)

(c)

(d)

vll

ii,

,/1//i/.~.///

/lJ

/../.

¢11

(e)

.,. ,,

(f)

(g)

~.

(h)

l

....~

/~iv.

(i)

Fie. 1. Schematic representation of various one- and two-photon spectroscopic processes. States m and n are the first and second vibrational levels of the ground electronic state and state r is the first excited electronic state. The horizontal wavy lines represent either incident or emitted photons. (a) Direct one-photon absorption resulting in vibrational excitation (where v,,, = iJ, - v,,). This corresponds to infrared absorption spectroscopy. (b) Direct one-photon absorption resulting in electronic excitation. This corresponds to visible-ultraviolet absorption spectroscopy. (c) Two-photon absorption resulting in electronic excitation (where ~,.,, = vl + i,2). (d) Direct one-photon absorption of a photon of energy hi, 0 followed, after a measurable time delay of about 10 r to 10-~° sec, by reemission of photons. This corresponds to fluorescence spectroscopy. (e) The virtual absorption and virtual emission (inseparable in time) of a photon of energy hv0. The extent to which energy> AE, is not conserved during the process (see text) is indicated by the dotted arrows. This corresponds to Rayleigh scattering. (f) "['he virtual absorption of a photon of frequency v = 1,0 and the ~irtual emission of a photon of frequency v = v0 - 9,,, resulting in vibl;ational excitation. This corresponds to Stokes vibrational Raman scattering. (g) The reverse of process (~ in which vibrational deexcitation occurs. This corresponds to anti-Stokes vibrational Raman scattering. (h) Preresonance Raman scattering--Raman scattering in which the energy of the incident photon almost matches that of an electronic transition. (i) Rigorous resonance Raman scattering--Raman scattering in which the energy of the incident photons matches the energy of an electronic transition. This is shnilar to a process referred to as resonance fuorescence (see text for details).

ever, according to the Heisenberg uncertainty principle in the form (AE)(At) >__ h, transitions in which energy is not conserved are possible provided that they take place in a sufficiently short period of time, At (= h/AE, where AE = E0 - Er), perhaps of the order of 10 -''~ sec in nonresonance scattering." Such transitions to (or from) excited electronic ~ W. L. Peticolas, Annu. Rev. Phys. Chem. 18, 233 (1967).

[S]

RAMAN AND RESONANCE

RAMAN SPECTROSCOPY

7]

states are referred to as " v i r t u a l " transitions. Since, in R scattering, there is no measurable time delay between the virtual absorption of the first photon and the virtual emission of the second, the molecule never really attains the intermediate state Er in this two-photon process, and hence the role of the intermediate state is purely virtual. For this reason, it is referred to as a virtual state. It will be seen shortly that the intensity of a nonresonance R transition is due to pairs of virtual absorptions and emissions involving a large number of virtual electronic states. In resonance scattering, the wavelength of the incident photons lies under the absorption band of an electronic transition. In such cases, the contribution to the scattering from those electronic state(s) with transition energies equal to that of the incident photons (i.e., the states with which the photons are in resonance) becomes very large and dominates all others. There results a selective enhancement in the intensities of vibrational transitions involving motions of those atoms about which the resonant electronic transition is localized. The particular vibrational modes enhanced are those that are coupled to the electronic transition responsible for the absorption band. The transition from R to rigorous rR scattering (in which E~ and Er are exactly equal) is a gradual one and traverses what is called the preresonance region. As rigorous resonance is approached, the lifetimes of the intermediate electronic states increase until, under the conditions of rigorous resonance, the distinctions between rR scattering and a process called resonance fluorescence (which can result in sharp Raman-like bands) become very subtle. Recently, the relationship between rR scattering and resonance fluorescence has been the subject of much attention, 7 ~'' and the reader should refer to the literature for further details. Under most circumstances, the two processes are clearly distinguishable since resonance fluorescence is a sequence of two independent one-photon processes separated by a measurable time delay. For this reason, resonance fluorescence is subject to quenching due to collisional deactivation during the lifetime of the excited state. Such theoretical subtleties, however, do not lead to practical difficulties for solid and liquid biological samples because their resonance fluorescence is generally quenched: their vibrational rR modes can, therefore, be recognized easily as discrete bands superimposed on the resulting, typically broad, fluorescence bands. The great practical advantages offered by the rR over the R technique are that (1) the enhancement in the intensities of the scattered photons ; D. L. R o u s s e a u and P. F. Williams, J. Chem. Phys. 64, 3519 (1976). P. F. Williams, D. L. R o u s s e a u , and S. H. Dworetsky~ Phys. Rev. Lett. 32, 196 (1974J. ~' S. M u k a m e l and J. Jortner, J. Chem. Phys. 62, 3609 11975). "~ J. Behringer, J. Raman Spectrosc. 2, 275 (1974).

72

CONFORMATION" OPTICAL SPECTROSCOPY

[5]

(i.e., the larger scattering cross section) allows the vibrational modes of chromophores to be studied at low concentrations; (2) the selective enhancement of only those vibrations associated with the chromophore leads to simple spectra, uncomplicated by the vibrations associated with the other parts of the molecule: and (3) the resonance technique can provide valuable information about the nature of the electronic transition with which the scattering is in resonance. On the other hand, the nature of the rR technique (in which chromophoric molecules are illuminated with intense light sources) often makes it necessary to have advance knowledge of the photochemistry and electronic spectroscopy of the sample in order to obtain and interpret the rR spectra. Theoretical Background In order to describe the properties of R scattering properly, to interpret R and rR spectra, and to provide a basis for further discussion, it is necessary to consider the theoretical basis of these effects briefly. Most of the topics discussed below will be applied in later sections. Classical Theory Existence o f a Vibrational R a m a n Effect

According to classical electromagnetic theory, when light comes into contact with a molecule, the electric field of the radiation, E, E = E' cos 2rrvot

(1)

(where v0 is the frequency, t is the time, and E' is the maximum amplitude) periodically disturbs its charge distribution, creating an induced dipole moment, p. The oscillating dipole that is formed radiates energy in the form of scattered light, the total intensity per second being given by I = (2(f'2))/3c 2

(2)

where (p2) is the time average of the square of the second time derivative of the induced electric moment and c is the speed of light. Rayleigh and R scattering can be accounted for by examining the frequency dependence of p. The quantity that describes the way in which the induced dipole moment is produced by the electric field is the polarizability, a, defined by p = a.E The polarizability is a tensor

(3)

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

73

{ O~'r3' Ot'~"~/ OL'rz t = ~j,~.

c~j

~j~

(4)

whose element a~p describes the induction of a dipole moment along the o- axis due to an electric field along the p axis, where o-,p = x, y, : in a l a b o r a t o r y - ~ e d coordinate system. The total induced moment in

direction (r is

P . = ~ ~,~oE~

(5)

p

It is the tensor properties of a that are responsible for the angular dependence of the intensity of the scattered light as well as its polarization. The polarizability is not a constant, but is subject to the periodic perturbation of the normal vibrational modes of the molecule. For small vibrations, the element ~,~p can be expanded in a Taylor series about the equilibrium nuclear configuration in terms of the displacements of the normal coordinates, ~Qk, of the molecule from its equilibrium configuration, as follows

~(Q~.) = ~po + ~ (oa~JOQDoQk + higher order terms

(6)

k

where km,~ = 3 N - 6 for nonlinear molecules and 3 N - 5 for linear molecules, ±Q~. is written as Qx for simplicity, and the zero superscript and subscript denote the equilibrium nuclear configuration at which ~Qk = 0. N o w , a~w(Q~.) contains an implicit frequency dependence, since the value of Q~. varies periodically as the molecule vibrates. If u~. is the frequency of vibration of the kth normal mode, one can write Qk = Q~.' cos 27rz,l,.t

(7)

where Q~ is the maximum value of Qk- Combining and rearranging Eqs. (1) and (5)-(7), the following expression is obtained for the dipole moment induced in direction op,, = ~ a~°E~, cos 27ruot P 1-+

+

+ cos 2.1,.,,-

, Itl

(st

-- o.L' The first term accounts for Rayleigh scattering, since the radiated light has the same frequency (u0) as the incident light. The second term provides the basis for anti-Stokes and Stokes R scattering at frequencies u,, + u~. and u0 - u~., respectively, for every normal mode k for which the differential polarizability (Oa~JOQ#)o is nonzero. The value of ~ ) then determines the scattered intensity according to Eq. (2).

74

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

The above criterion for R activity differs from that for infrared activity, where, to be active, the change in a normal coordinate must produce a change in the permanent dipole moment (Olx/OQ~.)o. For this reason, infrared and R spectra can provide complementary information since modes that produce strong bands in R spectra are often absent in infrared spectra and vice versa. For the biophysicist, a particularly important example of the difference between R and infrared spectroscopies is the application to the biological medium, water, which absorbs infrared radiation so strongly that only limited regions of the spectrum are available for the study of vibrations of solutes. Fortunately, water is a rather poor R scatterer, making R spectroscopy the technique of choice for aqueous samples.

Existence o f a Resonance Raman Effect In Eqs. (6)-(8), the dependence of c~ o on nuclear coordinates was developed classically to provide a basis for the existence of the vibrational R effect and a criterion for the R activity of normal modes. Now, to provide a classical basis for the existence of a rR effect, we consider the manner in which o~o varies with the wavelength of the incident light. For this purpose, we turn to the classical dispersion theory of dielectric media. According to this approach, when the electric field of an incident electromagnetic wave of frequency v0 interacts with an ensemble of particles of mass m and charge e, a damped, forced oscillation of the particles about their equilibrium positions results. The elements of the electronic polarizability tensor are, then, given by j~ e" , ~ (fr)~p a~° =47r2m l'i-2 -- /'0e + (1/27r)v0F,.

(9)

.

where f,.,/',., and F,. are the oscillator strengths, frequencies, and damping constants of the rth electronic oscillator. The oscillation of electric charge at frequency vr is identified with the absorption of electronic energy. When v0 approaches/'r for any r (i.e., when an electronic absorption band is approached), the absorption of energy by the dielectric increases greatly and the denominator of one term in the sum becomes very small, causing that term to become very large. This is referred to as "resonance." The large values of a [and (Oa/OQ)o, which also contains the resonance denominator] that result when/'0 approaches vr, give rise to resonance scattering. To predict which normal modes would undergo rR enhancement, Eqs. (9) and (8) would have to be considered together. This ~ J. Behringer, in "Molecular Spectroscopy" (R. F. Barrow, D. A. Long, and D. J. Millen, eds.) (Specialist Periodical Reports) Vol. 2, p. 100. The Chemical Society, London, 1974.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

75

question, however, is best dealt with by the quantum formulation that is given below.

Polarization of Raman Bands An important characteristic of a R band that must be understood to interpret R spectra properly is its depolarization ratio, p. This can be defined by reference to Fig. 2 in which a schematic diagram of a typical R scattering experiment is shown. The incident laser beam traveling along the x axis with its electric vector (E=) linearly polarized along the z axis impinges on the sample. The scattered light is usually observed at 90 ° to the incident beam--in this case, along the y axis. Depending upon the sample and the particular vibrational mode. the scattering process may "depolarize" the incident beam; i.e., light waves are produced whose electric vectors are perpendicular to those of the incident beam. The degree of depolarization is expressed as the depolarization ratio, p~. (where the subscript L denotes linearly polarized incident light), defined as shown in Fig. 2 by the ratio of the intensities of scattered light polarized perpendicular (I ~ ) and parallel (1H) to that of the incident light. I ~ and l, can be measured with the aid of an analyzer. The value of PL is used to characterize and help identify the type of vibrational mode responsible for a R band, and its use will be illustrated in later"sections where spectral interpretation is considered, The depolarization of the incident light is a consequence of the optical anisotropy of the sample. This anisotropy is described by the polarizabil-

I± Ix PL = -- = POLARIZATION ANALYZER

I~

x

Ix

Y

SCATTERINGOBSERVED / DIRECTION OF INCIDENT BEAM

9 0 ° TO INCIDENT BEAM

FIG. 2. Schematic representation of a typical Raman experiment defining the depolarization ratio, PL, of the scattered light when the incident light is linearly polarized.

76

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

ity tensor, a , and its elements can be related to pL- It should be recalled from Eqs. (6)-(8), however, that it is the polarizability derivative, (0a~o/ OQDo, that is responsible for R scattering. Therefore, it is actually the elements of the tensor o f the polarizability derivative that are related to the PL o f R bands. Below, however, PL will be expressed in terms of c~,p with the understanding that these are actually (Oa~p/OQA.)o. Only if the sample is a single crystal suitably oriented along its crystallographic axes with respect to the incident and scattered light can individual elements of a be related to I1 and I~. For fluids, the molecules are randomly oriented with respect to the laboratory-fixed coordinate system used to define a . Hence, when an average over all molecular orientations is taken, I i and IH are found to be related to certain tensor invariants (combinations of the various a~o). Accordingly, for randomly oriented molecules r' PL = (3g s + 5ga)/(lOg '' + 4g s) =11/111

(10)

where (lla) gS = ½ [(o~x~. - oz,,o)-' + (oz,.x- Olzz) z + (oz~j,v - O~zz)"-] + ½ [(c~x~ + Ol~z)" + (c~.,.~ + o~x)'-' + (c%~ + c~y) z] g " = ½ [(~.,., - o~,~)' + (o~x., - ,~.,.)' + {Cq, z - - ~ ) = ' ]

( 1 lb)

(llc)

Under ordinary circumstances (far from resonance), a is always symmetric (i.e., a~p = Up~) a n d g a = 0. The value OfpL can therefore vary from 0 to 3A. According to group theory, every normal mode can be assigned a given symmetry. The reader should refer elsewhere ~:~for a discussion of the application of group theory to molecular vibrations. It should be pointed out, however, that the symmetry of a normal mode limits the possible values of PL of its R band. For all modes that are not totally symmetric, g 0 = 0. Hence, far from resonance, the PL of a nontotally symmetric band is 3A, and such modes produce depolarized R bands. For all totally symmetric modes gO 4= 0 and these modes have values ofpL that range from 0 (strongly polarized) to 3/4, depending upon the relative values o f g ° and gS. N e a r resonance, ot can b e c o m e antisymmetric ( ~ -- -C~p~) '2G. Placzek, in "Rayleigh and Raman Scattering," UCRL Trans. No. 526(L) from "Handbuch der Radiologie" (E. Marx, ed.), VI, 2, 209 Akademische Verlagsgesellschaft, Leipzig, 1934. ,:3 E A. Cotton, "Chemical Applications of Group Theory." Wiley (Interscience) New York, 1963.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

77

for certain modes. This leads to anomalously polarized bands (i.e., bands with PL>3A). In the limiting case when g S = g O = 0, but g a # 0, the phenomenon of inverse polarization is encountered, in which pL = r_. The values of pL observed for R bands are useful in assigning symmetry species ':~ to them. Far from resonance, the relative values of the two tensor invariants, g" and g~, can always be determined from Eq. (10) by measurement of PL. When a is not symmetric, which can happen only near resonance, there are three tensor invariants which, in general, cannot be determined by measurement of PL alone. These three invariants, however, can be determined uniquely by also using circularly polarized incident light and analyzing the polarizations of the back-scattered or forward-scattered light, v-'.'4-1,; The classical theory outlined above is useful in that it provides an explanation for the existence of the R effect and yields the correct criterion for R activity. It fails, however, to provide any insight into the role of excited electronic states in the rR effect, nor does it suggest which vibrational modes undergo resonance enhancement. For this reason, it is necessary to examine briefly the key aspects of the quantum mechanical formulation of R intensities. Quantum Theory of Raman Intensities

Polarizability Theory Far from resonance, a quantum mechanical analog of the classical theory may be formulated. The quantum mechanical matrix element involving the a,,p component of the polarizability tensor for the transition from state m to n may be expressed as

(~o),,,,,

f to,,a~o+,,, dr =

where 0,,, and tO,, are the wavefunctions for states m and n. Now, in accordance with the polarizability theory of Placzek, 1"-'the Taylor series expansion of ~ o in terms of the normal coordinates of the molecule [Eq. (6)] is combined with Eq. (12) to give

(~o),,, =a~oO(n{rn) + ~., \(O-O-~e]oO, ] ,o. (,{Ot.{m> + higher order terms (13) In this expression, only matrix elements linking the initial (m) and final (n) states of the molecule appear. This represents a "ground state" approach ;4 W. M. McClain, J. Chem. Phys. 55, 2789 (1971). ':'M. Pezolet, L. A. Nafie, and W. L. Peticolas, J. Raman Spectrosc. 1,455 (1973). ";J. Nestor and T. G. Spiro, J. Raman Spectrosc. 1, 539 (1973).

78

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

to the quantum theory of R intensities because the role of the virtual excited electronic states discussed earlier is not considered explicitly. According to this polarizability theory, the intensities of R bands are related to the dependence of the polarizability of the ground electronic state on nuclear vibrations. This basic idea has been extended in bond polarizability theory ~7 to relate the effects of the bond stretching and distortion associated with each normal mode to its R intensities. Such approaches have proved to be of great value far from resonance, and it is for this reason that these approaches are mentioned here. To gain insight into the nature of resonance enhancement, however, one must turn to dispersion theory, in which the involvement of virtual excited states becomes more explicit.

Dispersion Theory Consider a molecule in molecular state m perturbed by a beam of electromagnetic waves of frequency u0 and intensityl0 causing a transition to state n and scattering light of frequencies u0 + v,,,,. For randomly oriented molecules, the total intensity of the scattered light is given by ~ 27~-5

I ..... - 3Zc~ lo(vo +- v,,,,,)4E

](~),,,,,1'-'

(14)

~rq)

When second-order time-dependent perturbation theory is applied to the interaction of radiation with matter, the well known K r a m e r s Heisenberg-Dirac dispersion equation [the quantum mechanical analog of Eq. (9)] for the elements of the polarizability tensor is obtained 1~1'

(~ro)mn=(1/h)E { (M°)r"(M~)"r + (M~),.,,(Me),,,,.] ,. v,.,, - v0 + iF,. vrn + v0 + iFrJ

(15)

where the sum over the index r covers all of the eigenstates of the molecule, h is Planck's constant, F,. is a damping constant which takes into account the finite lifetime and, hence, linewidth of each molecular state r [exp(-Frt) is the probability of finding the molecule in the rth excited state t seconds after excitation]. The (Mp'),.,,, etc.', are the amplitudes of the electric dipole transition moments defined as

(Mo)~,, = (n ]molr)

(16)

~7R. E. Hester, in "Raman Spectroscopy, Theory and Practice" (H. A. Szymanski, ed,), Vol. 1, Chap. 4. Plenum, New York, 1967. 15 j. Behringer, Z. Elektrochern 906 (1958). 19 j. Tang and A. C. Albrecht, in "Raman Spectroscopy, Theory and Practice" (H. A. Szymanski, ed.), Vol. 2, Chap. 2. Plenum, New York, 1970.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

79

where rnp is the electric dipole m o m e n t operator along direction p. The existence of a rR effect is immediately obvious from Eq. (15) for, as the excitation frequency, vo, a p p r o a c h e s that of an allowed molecular transition, v ...... the " r e s o n a n c e d e n o m i n a t o r " (v~,,,- v,~ + iF,.) b e c o m e s very small and the contribution of one term in the sum to the scattering bec o m e s very large and dominates all others. This denominator can, therefore, be thought of as a weighting factor. As resonance with a given molecular state is a p p r o a c h e d , there results a selective enhancement of certain molecular modes o v e r others. H o w e v e r , since Eq. ([5) does not distinguish electronic and vibrational states, the qualitative nature of the m o d e s being enhanced is not yet clear.

Vibronic Expansion Approach of Albrecht To this stage, the scattering has been treated as a problem involving molecular eigenstates only. The zeroth order B o r n - O p p e n h e i m e r approximation introduces the concept of separate electronic and vibrational states, and the role of vibrational perturbation of electronic states bec o m e s explicit in molecular s p e c t r o s c o p y . The electronic wavefunctions that pertain to the vibrationless molecule are the so-called zeroth-order electronic wavefunctions. In certain cases, electronic transitions that are forbidden by selection rules for the hypothetical vibrationless molecule are, in fact, actually found to be active in the real, vibrating molecule. The expression of this ~'forbidden intensity" is due to the mixing of the zeroth-order electronic wavefunctions of the ground and excited states by the vibrational motions of the molecules and is referred to as "~vibrationally induced intensity." In the same way that this mixing can impart activity into forbidden electronic transitions, it also can be shown to enhance the activity of allowed transitions. This e n h a n c e m e n t in intensity is referred to as the forbidden intensity in allowed electronic transitions. It is exactly this forbidden character in allowed electronic transitions that has been shown to be an important source of intensity of R bands. ''~~'~ The explicit role of the nuclear coordinates in R theory is introduced by treating the scattering as a problem in vibronic spectroscopy. This approach was first carried out by Albrecht, ~''~¢~ whose approach is outlined here. Other vibronic expansion theories have led to similar results.-'~ First, the zeroth-order B o r n - O p p e n h e i m e r approximation is introduced in which each molecular state is identified as a product of an electronic and a vibrational wavefunction. Thus m = ~qi. n = ,~,/, and r = ev, where ,~ is the ~ A. C. Albrecht, J. Chem, Phys. 34, 1476 (1961). e~ See Tang and Albrecht "~ for a s u m m a r y , and also W. L. Peticolas, L. A,Nafie, P. Stein, and B. Fanconi, J. Chem. Phys. 52, 1576 (1970).

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CONFORMATION:

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[5]

ground state, e an excited electronic state, and i, j are vibrational states associated with g, and v is a vibrational state associated with e. The corresponding wavefunctions are written [ m ) = [g)li)

I.> =

Ig) >

(57)

Ir>= le)lv>

where } ) signifies an electronic state (parameterized by nuclear coordinates) while ] ) is a vibrational wavefunction depending only on nuclear coordinates. Next, in accordance with Herzberg-Teller formalism, the electronic wavefunctions are developed in a Taylor series of nuclear displacements about the equilibrium nuclear configuration. Thus

le)- Ig")+ E/; ,vet Ec,,I,") le) = le") + E ~, c~,,.Is") #

(18) (19)

e--Ps

where the sum over k is over all normal modes and e, s, g, t refer to electronic states of the molecule. The coefficients ce.~ and c,, are evaluated by treating the nuclear motion as a perturbation and applying first-order perturbation theory using the zeroth-order wave functions Ig °) and Je") as the unperturbed functions and (OH/OQ~.)oQk as the perturbing operator, where H is the electronic Hamiltonian. One obtains (hk)e"°Qk ce,-- E o _ E.0

(20)

where E,/' and E.~" are the energies of states le °) and Is °) and

(hk)es ° = (e ° ]hk ]s °)

(2 l)

where hk, the vibronic coupling operator, is hk =

(22) 1)

with similar expressions for c , , When the Herzberg-Teller expanded electronic wavefunctions are introduced into the dispersion equation (15), and when the dependence of the electronic energies, E, on the nuclear coordinates is also taken into account, one obtains ''''-''' (dropping the sum over k and the damping constants, Fr, for simplicity) (c~p),i.,j =

(v~'-' - ~ - ='C.s

-

v.-)(v,-

-

vo")

o o o + ( M ~,oe~ ~ °th A.,~t ~ ° ' M ~).,.~ ~ ol1 × [(Mp)o~(h~.)~(M~).~o J~ (iJQ~.[j) e~ A. C. Albrecht and M. C. Hutley, J. Chem. Phys. 55, 4438 (1971).

(23)

[5]

RAMAN A N D RESONANCE RAMAN SPECTROSCOPY

81

It is useful to examine the significance of each of the quantities in this equation. The first quantity inside the sum describes the dependence of (c~,w) ..... on the frequency of the incident light. Again, it is apparent from the resonance denominators that, as the incident frequency (u,,) approaches the eigenfrequency of an allowed electronic transition (u, or ,<). resonance-enhanced scattering will occur. ( M , ) , : is the magnitude of the electric dipole transition moment between electronic states g" and e" along Cartesian coordinate P and similarly for the others. These moments describe the probability that transitions linking different electronic states will occur. The (h~.),,~" are vibronic coupling matrix elements which represent the perturbation energy per unit displacement of the kth normal mode resulting from vibrational mixing of the zeroth-order wavefunctions !e"t and Is"). These integrals (for e 7~ s) are a measure of the forbidden intensity in allowed electronic transitions discussed above. The quantity (M,),,?(hA.L.~'~(M~).~, ~ can be thought o f (following the terms from right to left) as describing a series of events in electronic space starting with a virtual dipole transition from electronic state Ig ~') to state Is") followed by vibrational mixing by hA. of virtual states le ~') and Is~') and then the transition from state le ~') back to Ig°). At the same time, vibrational states i a n d j of the ground electronic state are being coupled by Q , as found in the factor (ilQa.]j). On the basis of Eq. (23), two types of resonance processes may be distinguished which correspond to the cases e = s and e ¢ s and are referred to as A and B terms, respectively. Since e = s for A terms, one is dealing with a diagonal term o f the h~,. operator within a single excited electronic state. This is the more c o m m o n mechanism of resonance enhancement. Since h, has the same symmetry in electronic space as the normal mode k, the integral (h~.),,,/~ (of the A term) must vanish by symmetry unless k is a totally symmetric vibration. A-term resonance enhancement, therefore, leads to polarized R bands [see discussion of Eq. (10)]. The values of(h#)~ ~ are expected to be greatest for those vibrational modes that distort the molecule along the same coordinates as when the molecule goes from the ground to the excited state. Hence. such modes are expected to, and have been found -':~ to. undergo resonance enhancement. B terms involve vibronic mixing of two excited electronic states. Resonance enhancement arising from B terms is inherently weaker than that arising from A terms. The active vibrations may have any symmetry that is contained in the direct product of the group-theory representations ':~ of the two electronic states. From this B term of Albrecht's theory, there

'-': A. Y. Hirakawa and M. Tsuboi, Science 188, 359 (1975).

82

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

follows the prediction TM that those normal modes which are vibronically active in allowed electronic transitions should exhibit particularly striking e n h a n c e m e n t of their R intensities as resonance is approached. For this reason, rRS can supplement what is known from electronic spectra about vibrooic interactions in molecules. The intensity of an R band corresponding to a vibration that is vibronically active is predicted '-'4:-'~to have maxima at both the f r e q u e n c y of the resonant pure electronic transition, "e, and the f r e q u e n c y of the first vibronic transition, v,, __+A,w"'~ A- and B-term resonance-enhanced R bands may be distinguished by the frequency d e p e n d e n c e of their preresonance enhancement. By applying several simplifying approximations, Albrecht and Hutley-"-' have shown that, for A terms, I o~ (Vo +- viJ)4

[.e-' + .0-' -I-' (.-~'-Z .o2---))2J ~F4"-'

(24)

and for B terms

[

v~v, + Vo"_ I ~ 4(Vo - vi~)4 (v._, - ~o_,)~-~,.2_ .o2)

]-

=---Fd'-

(25)

where ,~j is the R frequency shift and I the intensity of the scattered light for the vibrational transition from i toj. These expressions can be used to help determine the m e c h a n i s m of the resonance enhancement. As a corollary of the above, it should be realized that, when obtaining rR spectra from a molecule with m o r e than one electronic absorption band, a different rR s p e c t r u m can be o b s e r v e d depending upon the choice o f exciting wavelength. Hence, the advantages to be gained by studying rR spectra as a function of excitation frequency cannot be overemphasized. The results derived in this section will be applied in a later section to the interpretation of the R and rR spectra of biological c h r o m o p h o r e s . H o w e v e r , first some of the experimental aspects of RS and rRS will be considered. Experimental Considerations The Raman Experiment R and rR spectra are obtained by analyzing the photons scattered inelastically (those for which v, -~ v~) from the sample. R spectra are ~4T. C. Strekas and T. G. Spiro, 2. Raman Spectrosc. 1,387 (1973). •~5j. A. Koningstein, J. Raman $pectrosc.. to be published, cited by Strekas and Spiro.~4 ~ This is also true for A-term resonance-enhancement. A model for the vibrational excitation profile of rR bands based on the vibronic approach has been developed recently (G. Korenowski and A. C. Albrecht, to be published).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

83

expressed as intensity versus frequency shift (~v = v~ - v,~,~,, or v., - v, in the nomenclature of Fig. 1) of the scattered photons (frequency v~) fiom the exciting laser line. In practice, 2xv is usually expressed in wavenumbers (cm r), where the wavenumber of a photon is defined as the reciprocal of its wavelength (l/X). The wavenumber and frequency differ by a factor of the speed of light, c, where v = c(1/,k)

(26)

Since c varies with the refractive index of the medium, values of ,k~,, expressed in cm -~, do also. For very careful work, ~v should be calculated in vacuum wavenumbers. '~ However, the differences between the values of ~u for air and vacuum are small and can usually be neglected. For Stokes scattering, Av is negative; for anti-Stokes scattering (which is weak and seldom used), 2~v is positive. For ease of comparison of Stokes R spectra with infrared spectra, and according to convention, e" l~t,I is displayed increasing from right to left. The frequency shift of a R band does not vary as it undergoes resonance enhancement; hence, corresponding bands in R and rR spectra have the same frequency shift. Furthermore, these are equal to the frequency of absorption of the corresponding infrared bands.'-'"' The useful parameters obtained for bands in R spectra are their (1) frequency shifts, (2) intensities, (3) depolarization ratios, and (4) band half-widths. Since fluorescence from the sample also produces inelastic photons at wavelengths similar to those of R scattering and because it is much stronger than R scattering, the R spectrum can easily be obscured by fluorescence. The major problem in RS is the removal of interference due to the fluorescence of impurities. In rR experiments, one will often observe fluorescence that is not produced by impurities, but is rather the natural fluorescence of the chromophore being examined. In such cases, the source of the fluorescence obviously cannot be removed without removing the rR scattering also. Experimental approaches for overcoming fluorescent interference in R and rR experiments are dealt with in a later section. ~ G. Strey, Spectrochim. Acta 25A, 163 (1%9). -'~ IUPAC R e c o m m e n d a t i o n s , Pure Appl. Chem. 36, 275 ~1973). e" It should be pointed out that, for polymers that adopt regular structures, the vibrational m o d e s of individual units in the chain can interact with each other. The frequency of a given mode (such as a C - - O stretching m o d e for a polypeptide) depends upon the phase relationship between the vibrating units that are interacting. Generally, of the many possible phase relationships, some are only infrared and some only R active. For this reason, it can appear that the infrared and the R frequency of a given mode for a polymer is not the same. In fact, however, the infrared and R b a n d s observed are different because they are due to different motions of the interacting units. Some of the phase-related m o d e s are both infrared and R active. In these cases, they have the s a m e frequency.

84

CONFORMATION" OPTICAL SPECTROSCOPY

[5]

While both infrared and R spectra provide information about the normal vibrational modes, the R technique enjoys a few distinct experimental advantages. As mentioned earlier, RS is much more useful for the study of aqueous solutions. Furthermore, the entire spectral range from 10 to 3000 cm -~ can be examined in a single scan on the same instrument whereas, in infrared spectroscopy, the far-infrared region is often hard to study. The polarization of the R scattering obtained from randomly oriented samples provides information not available from corresponding infrared spectra. Last, the existence of rR scattering as a special case of R scattering has no analog in infrared spectroscopy. Instrumentation The five basic components of the R spectrometer are the (1) laser, (2) sample compartment and associated optics, (3) monochromator, (4) photoelectric detection system, and (5) output device with options for data handling. R and rR spectra are obtained on the same instrument. Whether one assembles his own instrument or purchases a ready-made commercial one, there are certain features of each component that should be kept in mind. In general, the most important consideration in the choice of components is the spectral region that is to be studied (which is usually dictated by the sample). Some of the more important considerations for each component are mentioned below. More comprehensive discussions of instrumentation can be found elsewhere? ~' :~' Laser

Because they produce intense, well-collimated monochromatic beams of narrow bandwidth, lasers have become the standard excitation sources for R scattering experiments. A list of lasers that can be used for R excitation, compiled from data of Behringer:~"and Mooradian, :~:~is given in Table I. The choice of laser will depend principally on the sample. For obtaining R spectra far from resonance, any laser with a line of suitable power to which the sample is transparent is suitable, in principle. Most commercial spectrometers, however, are equipped with argon and/or krypton gas lasers, which are reliable and have intense lines in the blue, green, or red regions of the spectrum. In general, since the intensity of the :"' C. E. Hathaway, in "The Raman Effect" (A. Anderson, ed.), Vol. 1, Chap. 4. Dekker, New York, 1971. :~ S. K. Freeman, "Applications of Laser Raman Spectroscopy." Wiley, New York, 1974. :~'-J. ' Behringer, in "Molecular Spectroscopy" (R. F. Barrow, D. A. Long, and D. J. Millen, eds.), (Specialist Periodical Reports). Vol. 3, Chap. 3. The Chemical Society, London, 1975. :~:~A. Mooradian, Science 169, 20 (1970).

TABLE 1 LASERS USED FOR RAMAN EXCITATION"

Laser Argon gas

Krypton gas

A r g o n - k r y p t o n mixed gas A r g o n - k r y p t o n - x e n o n flowing gas

Xenon gas Neon gas H e l i u m - n e o n gas C a d m i u m gas Nitrogen flowing gas (pulsed) Tunable dye

Ruby Tunable gallium arsenide s e m i c o n d u c t o r injection

Wavelength ~' (nm)

Power" (mW)

351. I 20 363.8 20 454.5 100 457.9 250 465.8 100 472.7 15O 476.5 500 *488.0 1300 496.5 400 501.7 250 *514.5 1400 528.7 --'~ 350.7 100 356.4 5(1 476.2 100 482.5 20 520.8 70 530.9 200 *568.2 150 *647.1 500 676.4 120 799.3 100 Same as for argon + krypton ion gas lasers Same as for a r g o n - k r y p t o n gas ion laser plus 597.1 100 627.1 --'~ 871.6 3 969.8 10 332.4 50 *632.8 I00 325.0 15 *441.6 50 337.1 100 540.1 I0 From roughly 550 to 590 Roughly 100 for using sodium I-W p u m p fluorescein and 570 to power 625 using rhodamine 6G 694~3 500 837-905 10IX)

" The data in this table were compiled from J. Behringer, Chapter 3 in " M o l e c u l a r S p e c t r o s c o p y , " (R. F. Barrow, D. A. Long and D. J. Millen, eds.), Vol. 3 (Specialist Periodical Reports), The Chemical Society, London, 1975: and A. Mooradian, Science 169, 20 (1970). t, Only lines in the 300-1000 n m range are included. The lines most c o m m o n l y used for R a m a n excitation are indicated by the symbol * ' These n u m b e r s vary with the p u m p power and, hence, are only approximate. '~ Not listed in references cited in (a), but expected to be weak.

86

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

R effect is proportional to the fourth power of the excitation frequency [Eq. (14)], excitation at the blue end of the spectrum is more desirable. In practice, however, one is often forced to use excitation in the red to avoid interference from fluorescent impurities. To obtain rR spectra, a laser with a line that falls under an absorption band of the sample is required. Owing to the larger scattering cross sections in the rR effect, one can usually obtain rR spectra using lower power; hence, many of the weaker laser lines listed in Table I can be used. The ideal source for rR spectroscopy would be a tunable laser that would enable one to c h o o s e - a n y wavelength from the ultraviolet to the near-infrared. While visible excitation has been used in most rR experiments to date, increasing use is being made of ultraviolet excitation. There are several problems in this area, however, the most serious of which is the light source. There are near-ultraviolet lines in the argon, krypton, neon, nitrogen, and cadmium lasers that can be used for excitation. To obtain shorter wavelengths in the ultraviolet, the principle of second harmonic generation has been used to double the frequency of strong visible lines. For example, fi'equency doubling of the 514.5 nm line of an argon gas laser gives a few milliwatts of power at 257.3 rim. In addition to the problems associated with the acquisition of a suitable light source, an instrument set tip for use in the visible may not be satisfactory for ultraviolet work, since the latter requires quartz sample optics and a spectrometer with gratings and photoelectric detection that are efficient in this spectral region (next sections). It should be emphasized that, when working with lasers, caution should always be exercised to prevent eye damage. When aligning the sample in the laser beam, one is often subjected to beams reflected from the sample cell. To prevent eye damage, protective glasses should be worn. Such glasses are available commercially for use with laser light of any given wavelength.

Sample Compartment and Optics For a detailed discussion of different scattering geometries, collection optics, etc., the reader is referred elsewhere. :H:r''a~'a~ For routine work, a 90 ° scattering geometry (Fig. 2) is recommended. Only a few of the more practical aspects of the sample compartment and optics will be discussed here. When using a gas laser for excitation, it should be realized that, in :~ T. R. Gilson and P. J. Hendra, ~'LaserRaman Spectroscopy," Chap. 2. Wiley, New York, 1970. ::~'M. C. Tobin, "'Laser Raman Spectroscopy," Chap. 2, Wiley, New York, 1971.

[5]

RAMAN AND RESONANCE

RAMAN SPECTROSCOPY

87

addition to the major lasing line, the laser beam contains other weaker plasma lines of the gas. Rayleigh scattering of these lines from the sample can give the appearance of R bands, Hence, it is recommended that a narrow band interference filter, specific for the laser line in use, be placed between the laser and the sample. These filters can be obtained commercially for any of the major laser lines and have typical transmittances of about 50%. For samples in which these plasma lines will not obscure important spectral features, they may be used for frequency calibration. A list of the wavelengths of these lines can be found in the tables published by Striganov and Sventitsky. :~; For most samples, a capillary tube serves very well as a sample cell. However, when performing rR experiments, one may want to use a sample arrangement designed to avoid subjecting the sample to the detrimental effects of the radiation, such as heating, photolysis, or photoisomerization. For solids, this can be achieved by using a rotating sample cell :~ in which only a fraction of the sample is exposed to the beam at any given time, thereby minimizing the absorption of energy and essentially eliminating heating and decomposition. Molecular flow techniques :~':~' have been developed to accomplish the same purpose for liquid samples. In general, the light scattered by different vibrational modes has different polarizations. Unfortunately, these different polarizations are transmitted differently by the gratings and other parts of the monochromator, and can artificially distort the observed band intensities. To eliminate these effects, a polarization scrambler (a birefringent quartz wedge) can be placed just in front of the entrance slit of the monochromator to effectively randomize the polarizations of the scattered light. Monochromator

A double monochromator is usually used to analyze the wavelengths of the scattered light. For observation of small frequency shifts close to the exciting line (Au < 100 cm ') a third monochromator is sometimes used. The choice of gratings for the monochromator is extremely important. The efficiency of gratings is determined in large part by their blaze angle (the angle at which the grooves in the gratings are inclined to the surface). ~'~-~' Hence, gratings that have been "'blazed at 500 nm'" may not :~'~A. R. Striganov and N. S. Sventitsky, "Tables of Spectral Lines of Neutral and Ionized A t o m s . " Atomizdat, M o s c o w , 1966; English trans., Plenum, N e w York, 1968. ~;~W, Kiefer and H. J. Bernstein, Appl. Spectrosc. 25, 609 (1971). ::~ R. Mathies, A. R. Oseroff, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 73, I (19761. :~' R. H. Callender, A. Doukas, R. Crouch, and K. Nakanishi, Biochemistry 15, 1621 (1976). '" ' D i f f r a c t i o n Grating Handbook," Bausch and L o m b Diffraction Grating Research Laboratory, Bausch and L o m b , Inc., Rochester, N e w York, 1970. H "'Diffraction Gratings Ruled and Holographic," J and Y Diffraction Gl~tings, lnc., M e t u c h e n , New Jersey.

88

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

be suitable in first-order diffraction for work in the near ultraviolet or the red. Obviously, the efficiency of the gratings should be consistent with the wavelengths of the available excitation source(s) and the wavelength sensitivity of the phototube (next section). It should also be noted that gratings can have different coatings, which will also affect their efficiencies at different wavelengths. There are two types o f gratings in u s e - - r u l e d and holographic gratings. Ruled gratings have a few unusual properties that can lead to spectral anomalies. Because of periodic imperfections in their ruling, they can cause spurious spectral lines, called ~'ghosts" or "~satellites,'" to show up in R spectra. These may appear as false R bands. Since these effects are artifacts of the gratings, the spurious peaks can be seen even when there is no sample present in the sample cell--a fact that may be used to detect them. Furthermore, unlike R bands, the wavenumber shift of a ghost depends on the exciting wavelength and should change when the excitation frequency is varied. There is a variety of ghost, called a ~walking ghost, ''~-' that is particularly troublesome since its location in the spectrum is a function of both the wavelength and the angle of incidence of the light. Since it is the intense Rayleigh scattering from the sample that is responsible for ghosts, they can be reduced or eliminated by filtering out the Rayleigh light. The placement of a cutoff filter that transmits only photons with wavelengths longer than that of the laser beam between the sample and the monochromator will accomplish this. Holographic gratings are not ruled mechanically; instead, the grooves are produced by the effects of interference fringes on a photosensitive material deposited on an optically flat glass, As a result, they are essentially free of ghosts. It was mentioned earlier that gratings do not transmit different polarizations equally. In addition, for each polarization, the wavelength dependence of this transmittance is different and may exhibit unusual shapes referred to as Woods polarization anomalies.'" In general, holographic gratings have more serious polarization anomalies than ruled gratings. ~'' To compensate simultaneously for all the wavelength-dependent effects in a R spectrometer, the wavelength response of the entire instrument can be determined using a National Bureau of Standards lamp: frequency and intensity corrections can then be applied to the R and rR spectra obtained using any excitation wavelength. Photoelectrk" Detection Pulse counting photoelectric detection using a cooled phototube is now commonly used on most spectrometers. The important component in ~-'D. O. Landon and A. J. Mitteldorf, The Spex Speaker, XVII, No. I, 1972.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

89

such detection s y s t e m s is the phototube. Again, as with the other components, the wavelength of m a x i m u m sensitivity should match that of the other components. Phototubes that have a high sensitivity from the near ultraviolet all the way to the red are now available. For certain applications, however, a tube of very high sensitivity in a more narrow wavelength region may be desirable, An alternative to photoelectric detection that may be available in the future involves the use of light-amplifier tubes which act as "electronic photographic p l a t e s . " T h e s e would allow an entire R spectrum to be recorded in a matter of seconds, since no scanning by the m o n o c h r o m a t o r would be required.

Data Processing The signal f r o m the photodetection system can be converted to an output voltage and displayed on a chart recorder, the statistical fluctuations being smoothed electronically using a resistance-capacitance circuit. A more desirable alternative is to store the data in digital form on p a p e r or magnetic tape. The construction of a device for this purpose has been described recently. 4:~ The great advantage of such digital storage is that the raw data are stored permanently and may be displayed and processed over and over in any n u m b e r of independent ways. One can go a step further and interface a c o m p u t e r to control the workings of the entire spectrometer, as well as store, manipulate, and display data. T r e m e n d o u s strides have been made in this area, and the reader is referred to a recent review ~ on the subject. Sample Preparation The most important single concern in sample preparation is the removal of impurities that might absorb light and fluoresce, thereby obscuring the R or rR scattering of the sample. In cases where fluorescence is encountered, there are ways of adjusting the instrumental conditions to help bring out the R scattering a b o v e the fluorescence. H o w e v e r , in far too many cases these methods have been resorted to without first attempting to r e m o v e physically the source of the p r o b l e m - - t h e impurity. Purification is the method o f choice to overcome fluorescence. Usually, some form of recrystallization or c h r o m a t o g r a p h y will be successful. For many ~:~G. J. Perreault, R. E. Cookingham, J. P. Spoonhower, and A. Lewis, "Applied Spectroscopy," 30, 614 (1976). ~ J. R. Downey, Jr., and J. G. Janz, in "'Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, eds.), Vol. 1, p. I. Heyden, London, 1975.

90

CONFORMATION"

OPTICAL SPECTROSCOPY

[5]

macromolecules, treatment with activated charcoal, washing with sodium carbonate, or simple dialysis has given good results. Affinity chromatography would seem to be the best way to purify enzymes for R or rR studies. From the authors" own experiences, many enzyme preparations contain biological impurities, such as porphyrins or conjugated cofactors, which can absorb visible light and fluoresce. Besides producing intelfering fluorescence backgrounds, impurities may also give rise to rR scattering that can appeal together with the nom'esonance R scattering from the molecule under study. One can often determine in advance whether or not to expect fluorescence in R experiments through use of a fluorimeter. For aqueous solutions, one can obtain an emission spectrum using the wavelength corresponding to the laser wavelength. If no fluorescence bands appeal" close to the exciting line, and if the weak R band due to water (Au - 3500 cm ~) is visible, then there are no appreciable interfering fluorescent impurities present, and the sample should give a R spectrum. In cases where fluorescence bands appeal, they may help identify the impurities. For solid samples, crystals produce the best spectra. Protein crystals can be conveniently sealed in capillaries under their mother liquor to prevent dehydration and denaturation. The crystals should be small and randomly oriented with respect to the laser beam to avoid single-crystal polarization effects. If lyophilized powders are to be used, they should be packed down to obtain a high density and to prevent charring. Solid-phase spectra can also be obtained fiom samples suspended in solid matrices, such as KBr pellets or (at very low temperatures) in an inert gas matrix, Solution-phase samples should be free fi'om dust and undissolved material. Samples can be passed through a Millipore filter directly into a capillary tube with the aid of a syringe. A problem often encountered with concentrated protein solutions is the appearance of bubbles. Since these will result in a high degree of Rayleigh scattering and produce an unwanted background, solutions should be handled carefully to avoid foaming and bubble formation. It often helps to degas the sample before filling the capillary. With solution-phase samples, it is common to insert an internal frequency and intensity standard, such as SO4"-, which has a strong, polarized band at 981 c m - ' . ~:' To first order, the integrated area of a R band is proportional to the concentration of the species responsible for it. Because of an ~finternal field effect," ''; this is not strictly true, but is still usually a good enough assumption for biological samples. ~; G. Herzberg, "qnfrared and Raman Spectra." Van Nostrand-Reinhold, Princeton, New Jersey, 1945. ~" J. R. Nestor and E. R. Lippincott, .I. Raman Spectrosc. 1,305 (1973).

[,5]

RAMAN A N D RESONANCE

R A M A N SPECTROSCOPY

91

To obtain a nonresonance R s p e c t r u m f l o m a solution, one normally m a k e s the concentration as high as obtainable. In practice, it is difficult to obtain a good spectrum from solutions of concentration less than about 2 w / v ~ . One disadvantage of RS that is usually not emphasized is that relatively high concentrations are required. One must be aware that intermolecular effects can influence the spectra. Hence, whenever possible, spectra should be obtained over a range of concentrations to see whether differences due to such effects are encountered. If valid conclusions regarding the conformation, etc., of proteins are to be drawn from R spectra and discussed in relation to information obtained from other sources, the experimental conditions (e,g., concentration, solvent, temperaturel under which tile various experiments were carried out should be the same or the differences between them shown not to matter. RS of proteins is generally carried out at concentrations higher than with many other techniques, such as infrared, ultraviolet-visible, circular dichroism spectroscopies: rR s p e c t r o s c o p y , however, does not suffer from this disadvantage. The optimization of concentration for rR experiments is more critical than for R experiments. Both the incident and scattered light are absorbed by the sample. The absorption of light from the laser b e a m leads to a "'thermal lens effect." The absorbed light causes a local rise in temperature that results in a refractive index gradient in the absorbing medium. This gradient acts optically as a "'thermal lens'" and will cause divergence of the beam. This divergence may result in a change in the power density of the incident beam in the volume element of the sample from which the scattered light is being analyzed. In addition, before starting the R experiment, one should wait until thermal equilibrium (and therefore steady power density) is achieved, in order that the focus of the beam not be disturbed. In Fig. 3, the absorption s p e c t r u m of a hypothetical c h r o m o p h o r e is shown. The arrow indicates the position of the 514.5 nm line of an argon laser and the brackets indicate the region in which the 500 to 2000 cm Stokes rR s p e c t r u m falls, it is obvious both that the scattered light will be absorbed by the sample and that the extinction coefficient (ca) will depend on the fl-equency shift of the photon. To minimize the absorption of both the incident and scattered light, a transverse excitation geometry is reco m m e n d e d . This is illustrated in Fig. 4. The advantage of transverse excitation is that the pathlength of the incident and scattered light through the sample is short. For some samples, the use of a focused b e a m will tend to result in sample decomposition, even at low powers, and an unfocused beam must be used. The self-absorption of scattered light can be minimized by positioning the focus of the laser b e a m close to the side of

92

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

Stokes / Roman Spectrum

Ulaser 514.5

/

z~v = 5 0 0 - 2000 cm- I

-.--II I I •

',

"_x_ .........

450

500

550

~(nm) FIG. 3. Illustration of the wavelength region in which the Stokes resonance Raman scattering from an arbitrary chromophore, due to excitation with the 514.5 nm laser line, is observed.

~

pillory

Scattered

•

Light

r

Laser Beam

FIG. 4. Illustration of a Raman experiment using transverse excitation geometry with a focused laser beam; i.e., the axis of the capillary tube, the laser beam, and the observed scattered beam are all mutually perpendicular.

[5]

RAMAN AND RESONANCE

RAMAN SPECTROSCOPY

93

the capillary, but this is difficult in practice. Obviously, it helps to use capillaries with small internal radii (r), usually less than ~ 1 ram. The intensity of the light scattering increases approximately linearly with the concentration of the scattering centers. The absorption of the scattered light, however, increases exponentially as a function of sample concentration [see Eq. (27)]. As a result, there is an optimal concentration at which maximal scattering will be observed outside the sample cell. The question of optimal sample concentration for transverse excitation with a focused laser beam has been examined by Strekas et al.;r By assuming point scattering from the focus of the beam, the authors conclude that the optimum concentration corresponds to an absorbance (per 1 cm pathlength) of 13. This is r e c o m m e n d e d as a starting concentration for first observing a rR effect. One should, however, vary the concentration in the neighborhood o f this value to optimize it for his particular setup.

Correction of Resonance Raman Spectra for Self-Absorption The observed intensity, lob~, of a band at wavelength ~ in a rR spectrum (see Fig. 3) is given by

i,,~,s = Ixexp ( - 2.303rce~)

(27)

where Ix is the intrinsic scattering intensity at the focus of the beam, r is the scattering path length through the sample, c is the molar concentration of chromophore, and ex is the molar absorptivity at X. The correction to be applied for self-absorption is made relative to an arbitrary reference band in the spectrum. The observed intensity ratio, R,,~,~, of any two bands is given by Rob.~ --- R xe e':~":~''-x~,

(28)

where Aex is the difference in their molar absorptivities and R is the "'true" intrinsic intensity ratio. Then log Roi,s = log Rx - rcAe~

(29j

By obtaining rR spectra as a function of concentration, one can plot log R,,~,,~ vs cAex to obtain a straight line whose slope is - r . Using this value of r as an "effective path length" for the scattering experiment, R~, the corrected intensity ratio, can be calculated at any given sample concentration for any band, using Eq. (28). ~ T. C. Strekas, D. H. A d a m s , A. J. Packer, and T. G. Spiro,

Appl. Spectrosc.28,324 (1974).

94

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

Choice of Experimental Conditions To obtain a nonresonance R spectrum of a sample showing fluorescence which, for one reason or another, cannot be removed physically from the sample, three approaches can be taken. One should first try to vary the excitation wavelength to reduce the absorption of the impurity, and thus circumvent the fluorescence problem. In general, though not always, excitation further into the red will reduce the fluorescence. Alternatively, most fluorescence backgrounds can be reduced by " b l e a c h i n g " the sample--i.e., by prolonged exposure to the laser beam. This may selectively photolyze the impurities responsible for the fluorescence. While this approach is used frequently, it involves some risk of damage to the sample and is not the method of choice. Finally, since fluorescent emission is sensitive to environmental factors, one can try various quenching procedures such as changing the phase, solvent, temperature, etc., or try adding a quenching agent (e.g., nitrobenzene:~"). In some cases, if the fluorescence is not too strong, the R spectrum can be detected as sharp peaks superimposed upon a broad fluorescence background. In theory, R scattering and fluorescence can be separated experimentally by time discrimination since R scattering occurs at least 100 times faster than the fluorescence. Experimentally, this would require the use of a pulsed laser and time-adjusted gate electronic detection. At least one such study 4~ has been reported, and it may provide a spectroscopic solution to the nagging problem of fluorescent interference. Another technique has been developed recently 4:~'5° in which R scattering can be separated easily from interfering fluorescence. It is called coherent anti-Stokes Raman spectroscopy (CARS). In this technique, a sample is subjected simultaneously to two crossed, focused, high-intensity laser beams, one of which is tunable. An anti-Stokes R signal is produced in the form of a spatially coherent beam from which interfering fluorescence can be easily filtered. Laser power is measured with a power meter and is usually adjusted to any convenient level, typically 5 to 300 milliwatts (roW), the upper limit being determined by what the sample will tolerate. The laser power used to obtain a rR spectrum is a critical parameter and should be chosen carefully. In general, lower powers are used for rR than for R experiments, typically between 10 and 50 roW. Since the absorption of laser light may cause heating and photodegradation or isomerization, it should be shown by some criterion that sample degradation does not occur. 4~R. P. Van Duyne, D. L. Jeanmaire, and D. F. Shriver, Anal. Chem. 46, 213 (1974). 4~'R. F. Beg|ey, A. B. Harvey, R. L. Byer, and B. S. Hudson,Am. Lab. 6, ll (1974). ~"R. F. Begley, A. B. Harvey, R. L. Byer, and B. S. Hudson, J. Chem. Phys. 61, 2466 (1974).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

95

Whenever possible, in any type of R experiment, the activity or some other critical property of a biological sample should be checked before and after exposure to the laser beam. Spectra taken consecutively on the same sample should be reproducible. Because o f the high power density at the focus of a laser beam, even apparently nonabsorbing samples can char. Hence, one should try to use the lowest power at which a highquality spectrum can be obtained conveniently. For rR spectra, the choice of wavelength should be guided by the absorption spectrum. When there is a choice of different excitation frequencies, each should be examined and the best determined by trial and error. The degree of resonance enhancement can be determined conveniently by comparing the band intensities of the sample with that of a nonresonant internal standard. A plot o f the degree o f resonance enhancement versus wavelength is called an excitatkm profile and can provide valuable information about the chromophore, as will be seen shortly. Other instrumental variables, such as scan speed, time constant (for chart recording), etc., should be adjusted to maximize the signal-to-noise ratio. Most commercial instruments list guidelines for these variables. In practice, most of the problems associated with obtaining good R and rR spectra from nonfluorescent samples arise from sample alignment and the collection and transfer of the scattered light to the entrance slit of the monochromator. Sometimes, it may seem as though a sample will not produce R scattering. If this occurs, one should first realign the sample holder and collection optics using a sample that is a strong R scatterer (e.g., benzene, carbon tetrachloride). One can then try to detect the weaker R scattering due to air (a convenient band 5~ is that at 61 cm 1). Having done this, one can attempt to observe the R scattering of the original sample, knowing that only minor adjustments in sample alignment should be necessary. Raman Spectral Artifacts As in any spectroscopy, when obtaining and interpreting R and rR spectra, one must be aware o f the possible artifacts that can lead to false spectral features. Here we summarize briefly some of the possible sources of trouble against which to guard. Extra bands (i.e., those not due to the sample) in a R spectrum may arise from grating ghosts, satellites, or laser plasma lines. These are often recognized as being unusually sharp or intense relative to the rest of the :'~G. Herzberg, "'Spectra of Diatomic Molecules," p. 62. Van Nostrand-Reinhold, Princeton, New Jersey, 1950,

96

CONFORMATION" OPTICAL SPECTROSCOPY

[5]

spectrum. If the laser beam is not focused carefully on the sample, it is possible that R scattering due to air can appear in the spectrum. This can contribute peaks in the region below about 150 cm -~ (due to the rotational transitions of 02 and N.,) as well as at 1554 and 2309 cm -~ (due to the stretching modes of O., and N2, respectively)? ~One should also be careful to carry out R experiments in a darkened room. Should any stray light-say, from a nearby fluorescent lamp--permeate the entrance slit, spurious peaks can result. The possibility always exists in R spectroscopy that the laser beam will damage the sample. Hence, one should always make sure that the R scattering of degradation products is not being observed. A commonly encountered phenomenon in R spectroscopy is a steady decrease in the overall signal measured during the course of a scan (especially a long one). This may be due to the fact that the instrument responds differently at different wavelengths. If so, appropriate corrections (based on a previous calibration with a standard lamp) should be applied. Often, however, this may result either from a drift in laser power, a bleaching of the background fluorescence due to impurities, sample degradation, or changes due to sample heating, such as thermal lens effects, which may cause deviations in the power density in the scattering volume. All of these time-dependent factors can give the appearance of true intensity changes, and caution should be used in interpreting such effects. For example, when measuring values of PL for bands, the _1_ and [[ scans should be taken one after the other, as quickly as possible, over small wavelength regions at a time, to avoid errors due to such drifts in intensity. When using R spectra to monitor time-dependent changes, such as protein denaturation, the same caution should be applied. Internal standards are useful for separating such drifts in total scattering intensity from true intensity changes. Finally, when measuring values of PL, one should make sure that the direction and polarization of the incident beam is not perturbed by reflections or refractions from the sample cell or the sample itself. The value of PL for the scattered light is useful only when the direction and polarization of the incident light is well defined. When knowledge of the incident direction or polarization is lost, PL can no longer be interpreted in terms of Eqs. (10) and (11). For example, it is not possible to obtain a meaningful value of PL for a finely divided crystalline powder, even though the molecules may be considered randomly oriented, since multiple reflections of the incident beam from the polycrystalline surfaces will scramble its polarization. Chromophores for Resonance Raman Experiments To obtain rR spectra, one must have a chromophore that absorbs in a spectral region that can be studied conveniently with available excitation

[5]

R A M A N A N D RESONANCE

R A M A N SPECTROSCOPY

97

wavelengths. This requirement is presently satisfied best by a chromophore that absorbs visible light although, as mentioned earlier, ultraviolet excitation shows promise for the near future. It is also desirable to be able to excite under an isolated absorption band of the molecule so that the probability of observing simultaneous rR scattering from more than one transition (which may complicate interpretation) is remote. In addition, the chromophore should not photolyze, photoisomerize, etc., when exposed to the laser beam, nor should it fluoresce. At the present time, there are basically two approaches to the rR study of biological molecules with visible excitation. One is to study molecules that are inherently chromophoric, such as heme proteins, certain nonheme metalloenzymes, and visual pigments. The other approach is to attach a chromophoric "resonance Raman label" to an inherently colorless molecule. One can then hope to use the rR spectrum of the label to reflect changes that can be related to its environment. The effectiveness of this labeling approach lies in the design of the label. The key consideration is to achieve specific labeling at sites of interest--usually those related to the biological function of the molecule. For example, for enzymes one might design chromophoric substrates or competitive inhibitors that, under a given set of conditions, are known to reside at the active site. Since the majority of the biological molecules of interest do not absorb in the visible, their access to study by rR spectroscopy will depend upon how cleverly suitable probe molecules can be designed. In the next sections, examples of each of the two major approaches mentioned above will be examined. Interpretation of Spectra In this section, the interpretation of R and rR spectra of proteins is considered. In particular, the types of information that can be obtained will be reviewed and, with the use of illustrative examples, attention will be called to those spectral features that have proved particularly fruitful for study, General Approach to the Interpretation of Raman Spectra of Proteins R spectra provide information about the vibrational motions of molecules. Since these vibrational motions are sensitive to molecular conformation and environment, they can be used to obtain information about the three-dimensional structure of proteins. The great advantage of vibrational spectroscopy over the various forms of electronic spectroscopy (ultraviolet-visible, circular dichroism, fluorescence, etc.) is the discrete nature of the spectra, which contain many bands of different origin that can be studied simultaneously.

98

C O N F O R M A T I O NOPTICAL ; SPECTROSCOPY

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As in infrared spectroscopy, the origins of the various bands can be established by using the " g r o u p - m o d e " concept, according to which certain groups of atoms are consistently found to be responsible for bands in certain narrow regions o f the spectrum, regardless o f the molecules in which they appear. For example, the CH stretching motions of aliphatic molecules produce bands in the 2800-3000 cm -j region3 '~ Hence, one of the most important steps in the interpretation of R spectra is the use of the group-mode concept to identify the bands of interest with the motions of certain groups o f atoms. In doing so, the origins of bands may be traced to very specific regions of the protein, and, hence, R spectra can provide information about local protein conformation. R spectra are used to obtain information in primarily two ways. The first and most frequently used approach is to correlate the frequencies of bands of known origin with specific conformations. This approach requires frequency-conformation correlations, which have been established previously (usually from studies on model compounds). The other approach, used when specific frequency-conformation correlations have not been established, is a phenomenological one in which observed spectral changes induced by protein denaturation, change of phase (e.g., from crystal to solution), etc., are used to study changes in conformation, without being able to identify their origin. Examples of each of these approaches are considered in the following sections. Far from resonance, all the active vibrations are observed and one is presented with a rather complex spectrum. In spite of this complexity, specific information can be obtained from the analysis o f certain spectral regions, as will be seen below. In rRS, one sacrifices scope for simplicity, and obtains information only about those atoms in the immediate vicinity of the chromophore. For convenience, R and rR spectra are discussed separately. Protein Conformation A protein backbone (see Fig. 5) consists of a series of planar trans (co = 180°) amide units (CONH) which are separated by single bonds from F . . . . . .

I c¢

R

--.n

i I

f0

L ......

[- . . . . . . .

HI

j

(l

CH 2

X,2e, l..,

I

I0

L ......

e.

c'v

R

0

v

,:.v

"

J

OH

FIG. 5. A section of a polypeptide chain illustrating the backbone dihedral angles 4', tO, and co and the side-chain dihedral angles X~and X~ for the serine and proline residues. The amide groups are all in the planar trans conformation.

[5]

R A M A N A N D RESONANCE R A M A N SPECTROSCOPY

99

a-carbon atoms. The various side chains are also connected by single bonds to the a-carbons. The conformation of a protein is determined largely by rotation about the single bonds of the main chain (described by the dihedral angles + and t~) and the side chains (described by the dihedral angles ×1, X", etc,). A residue, R~, is defined by the [NH-C~HR~-C'O] unit, to which the dihedral angles 4)~, t~, pertain. In Fig. 5 is shown a section of a polypeptide chain containing a serine residue, R~, and proline residue, R~.._,. The bond lengths and bond angles of the various atomic groupings also show some variation, but are confined, for the most part, to rather narrow ranges and, hence, do not influence the conformation significantly. In contrast to the other naturally occurring amino acids, the proline residue has a unique structure in which the C ~ and peptide N atonas are part of a five-membered pyrrolidine ring. As a result, the peptide unit of a proline residue has no amide hydrogen. Also, whereas the trans conformation (~o = 180°) is preferred for nonproline residues, the peptide group preceding the pyrrolidine ring of proline can adopt either a cis (oJ = 0°) or trans conformation (see Zimmerman and Scheraga :'~ for the interactions that lead to these preferences). Furthermore, rotation about the N--C" bond is severely restricted. Rotation about the various single bonds of the other types of residues is also not free, and there are certain preferred sets of values of d~, + adopted by the backbone and of )~', )(-', etc., by the side chains of the amino acid residues of proteins. :':~ The frequency-conformation correlation approach will be developed separately for the backbone and side chains. For this purpose, the vibrational modes that, in practice, are found to be useful for study are identified. Then, a systematic definition of those conformations that might be expected to be distinguishable on the basis of their vibrational frequencies is formulated. Finally, the results of studies of model compounds are used to correlate the frequencies of various bands with the specific conformations discussed. The frequency-conformation approach formulated here for the protein backbone differs from those commonly employed in the recent literature: a discussion of the differences between these approaches will also follow. Nonresonance Raman Spectra of Proteins Conformationally Sensitive Backbone Vibrational Modes The vibrational frequencies of the protein backbone can be divided into internal amide group modes and skeletal stretching and bending :'~ S. S. Zirnmerman and H. A. Scheraga, Macrornolecules 9, 408 (1976). '~:~S. S. Zimmerman, M. S. Pottle, G. Nemethy, and H. A. Scheraga, Macromolecules, 10, 1 (1977).

100

CONFORMATION: OPTICAL SPECTROSCOPY

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TABLE II VIBRATIONAL MODES OF THE PLANAR TRANS AMIDE (CONH) GROUP"

Approximate frequency r' (cm- ~) Designation Amide Amide Amide Amide Amide Amide Amide Amide Amide

A B 1 II Ill IV V VI VII

CONH

COND

3280 3090 1653 1567 1299 627 725 61111 206

--" --' 1642 1475 960 627 510 (627) --"

Description ~N--H stretching Fermi resonance" [2 × Amide 11 C==O stretching N - - H in-plane bending. C - - N stretching'~ C - - N stretching, N--H in-plane bending" O==C--N in-plane bending N - - H out-of-plane bending C==O out-of-plane bending C--N torsion

" From data given in T. Miyazawa, in "'Polyamino Acids, Polypeptides and Proteins" (M. Stahmann, ed.), p. 201, Univ. of Wisconsin Press, Madison, Wisconsin, 1962. ~' These modes are from the model compound N-methyl acetamide and its N-deuterated analog. " Not listed by Miyazawa." ,i This refers to a resonance interaction between the N - - H stretching mode and the overtone of the Amide I1 mode, which are accidentally degenerate, producing the Amide A and B bands. " The amide I1 mode has more N - - H bending character than does the amide 111 mode (see Table 18.2 of Miyazawa").

modes of the N - - C ~ - - C ' unit. The predominantly local amide group vibrational motions of each unit in the chain and the approximate frequencies of their R bands have been derived from the model compound N - m e t h y l a c e t a m i d e ) 4 and are listed in Table II. The amide I and amide III bands are easily recognized, and are fairly good group-modes because they lie close to the frequencies listed. Furthermore, their frequencies are sensitive to the local backbone conformation. Hence, these two modes have proved to be the most useful for frequency-conformation correlations. In polypeptides, the frequencies of amide I modes fall in the 16451675 cm ~ region and of amide Ill modes in the 1230-1295 region? The other amide group modes are less easily recognized and are too sensitive to conformational changes to be useful. The intensities of the skeletal stretching modes of the N - - C ~ - - C ' unit, v(NC~C'), have also proved to be useful and lie in the 870-1150 cm region? More specifically, the u(C~--C ') and t,(N--C ~) modes lie in the :" T. Miyazawa. in, "'Polyamino Acids, Polypeptides and Proteins" (M. Stahmann, ed.), p. 201. Univ. of Wisconsin Press, Madison, Wisconsin, 1962.

[5]

R A M A N A N D R E S O N A N C E R A M A N SPECTROSCOPY

101

870-950 and 1040-1150 cm -1 regions, respectively. However, since these two skeletal stretching motions are probably at least weakly coupled to each other, we prefer to refer to them collectively as u(NC~C ') modes. The vibrational modes listed in Table II are not expected to apply rigorously to proline amide units, since they are part of a ring and have different structures. The vibrational modes of proline amide units (which are trans in globular proteins) will not be dealt with here, but it should be noted that R spectra of poly-L-proline chains with cis (poly-L-proline I)~'~'~t~and trans (poly-L-proline II) 5~'~7"~8peptide units have been obtained and each of the two forms has characteristic bands. We will now describe a model in which the backbone vibrational frequencies of the amino acid residues in proteins and synthetic polypeptides (except proline) are considered to be determined by three factors. The first is the value of ~b,+?:' Variations in these dihedral angles can lead to changes in vibrational coupling between adjacent amide units bonded to the same c~-carbon. These changes in vibrational coupling, in turn, can change the amide vibrational frequencies. The second factor is the nature of the amide hydrogen bonding. To keep the model relatively simple, every amide group is assumed to be either hydrogen-bonded to another amide group within a local topographical structure (such as an c~-helical, parallel, or antiparallel fl-pleated sheet structure) or hydrogen-bonded to water (solvated). Since amide-amide and amide-water hydrogen bonds have different strengths, they should perturb the amide vibrational frequencies to different extents. Hydrogen bonding between amide groups and polar side chains will be neglected. Third, within the different topographical structures possible, interactions between nearby amide units (such as dipole coupling6°'"t) may introduce additional perturbations of the amide vibrational frequencies. Perturbation calculations~.~°.'" have been carried out for such regular structures as the c~-helix and the parallel and antiparallel pleated sheet structures. The results indicate that the R active vibrations of amide units in these infinitely long, perfectly formed structures can vary considerably, and such variations in the amide I and III vibrational frequencies have '"' W. B. Rippon, J. L. Koenig, and A. G. Walton, J. Am. Chem. Soc. 92, 7455 (1970). :"; A. M. Dwivedi and V. D. Gupta, Chem. Phys. Lett. 16, 109 (1972). ;7 M. Smith, A. G. Walton, and J. L. Koenig, Biopolymers 8, 173 (1969). :'~ V. D. Gupta, R. D. Singh, and A. M. Dwivedi, Biopolymers 12, 1377 (1973). "' In a recent study, S. L. Hsu, W. H. Moore, and S. Krimm,Biopolymers 15, 1513 (1976), the effects of variations of the values of ~5, + on the amide I and Ill vibrational frequencies of N-acetyI-L-alanine N - m e t h y l a m i d e have been investigated using a normal mode analysis. '"' S. K r i m m and Y. Abe, Proc. Natl. Acad. Sci. U.S.A. 69, 2788 (1972). ';' W. H. Moore and S. K r i m m , Proc. Natl. Acad. Sci. U.S.A. 72, 4933 (1975).

]02

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

been observed.:' The net result of these three factors is that many of the amide vibrational frequencies of amino acid residues that are part of the different structures found in proteins have unique and characteristic values. Criteria will now be established to define these different structures on the basis of their values of 6,tO and their states of hydrogen bonding. Because of the simplifying assumptions of the above model, the number of possible types of backbone conformations whose amide groups might be expected to have distinct vibrational frequencies will be underestimated. However, as will be seen shortly, even the above simple model leads to many more types of conformations than we are presently able to detect in R spectra of proteins.

Backbone Con3~rmational States, Topographical Structm'es, and Topographical States To define the different types of conformations o f residues that might be expected to have distinct backbone vibrational frequencies, residues are first grouped solely on the basis of their values of d~,t0. In this way, each residue is assigned to a conformational state. Because of restricted rotation about the N - - C ~ and C~--C ' bonds of the protein backbone, the frequencies of occurrence of various values of 6 , 0 are found to cluster in certain characteristic regions. This is illustrated in Fig. 6, in which the distribution of occurrences of values of dJ,$ for all the alanine, glycine, and proline residues in 29 proteins ~;~ is shown. The plot shown for alanine is characteristic of all o f the other amino acids except glycine and proline, which are shown separately for this reason. Residues whose values of 6,~0 fie in the regions designated ~R, O~L,and • , are in the right-handed c~-helical, left-handed c~-helical, and extended conformations, respectively. The regions between o~R, O~L,and • are referred to as the right- and left-handed bridge conformations (designated ~R and ~e, respectively). Because glycine has only a hydrogen atom for a side chain, its range of accessible values of 6 , 0 is much larger than for any other amino acid. For this reason, its amide group vibrational frequencies may be expected to vary more widely than those of the other amino acids. ,~2The data were compiled from the X-ray structures of bovine pancreatic trypsin inhibitor. carp muscle calcium-binding paralbumin, the A. B, and C chains of tosyl-~-chymotrypsin, concanavalin A. carboxypeptidase A, chymotrypsinogen A, cytochromes b~ and c2, tosyl elastase, clostridial flavodoxin, D-glyceraldehyde-3-phosphate dehydrogenase, the A and B chains of human deoxyhemoglobin and horse methemoglobin, sea lamprey hemoglobin, lactate dehydrogenase, oxidized chromatium high-potential icon protein, dogfish lactate dehydrogenase, hen egg-white lysozyme, sperm whale myoglobin, papain, ribonuclease S, rubredoxin, staphylococcal nuclease, subtilisin BPN, thermolysin, and trypsin.

[5]

RAMAN A N D RESONANCE RAMAN SPECTROSCOPY -180 180 1:5

0 I

IZ

I

I~IS

t80

1

,I

i I I '; i 2Z24

21211214121

I

103

A L A

L i L

(1 L i

q

i !

i ~ i

i ]

i ]

{

i

i i IeZGBG2

~ZR

~ i i

, ~L

,,3~4, i 22~1

i i

-180 ii 180 , ~ '

i

i

i

I1~

[

I I

~,~,,

,

t

1

i

~ , ~,,

.

i, '~,

121 I

'

2

i ,,2: I

I

i

I

2

i i

i

~R

ii,, z

,

E

0-I

q

" 1,33S2

i

i

Is~4 21~4z

i 1211 1211 Srr

IZ

~R ~''"~ 2 23413

-9-

iz 1121

2

~L

~224221

2 i

21 22

-180 180

i

I i

*

i

,

1

,~

2112

I

I

[

I

~L ,f

i

~L

I

142~2 23?2

P R 0

~

12 2 ~24~1

0

~,,I

111211

~

~R

~

i

I

4~,I 2~'~,,

ii

I1~

ii

i

II

i i i

i

ll!~ i , :

I

,

I I I

I I

i

~R I

O/R

E

L

E

J

-180 L~ -180

1

i80 (degrees)

FIG. 6. Distribution of dihedral of 32 proteins. The Greek symbols basis of their characteristic values rences. The symbols A through K

angles (b,~b for the alanine, glycine, and proline residues denote the different conformational states defined on the of (b,¢, and the numerals indicate the number of occurrepresent 10 through 20, respectively,

104

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

T A B L E III APPROXIMATE DIHEDRAL ANGLES FOR SOME REGULAR STRUCTURES"

aRn O~UJ ep GAp Poly-L-proline I Poly-L-proline II " From IUPAC--IUB 3471 (1970).

(deg)

(deg)

(deg)

--57 +57 --119 139 -83 -78

--47 +47 +113 + 135 + 158 + 149

+ 180 + 180 +180 - 178 0 + 180

-

-

C o m m i s s i o n on Biochemical N o m e n c l a t u r e , Biochemistry 9,

This fact should not be neglected, since glycine comprises about 9% of all the amino acids in globular proteins. Proline, in contrast to glycine, has a very restricted range of values of +,tO. For this reason, and since it cannot participate in hydrogen bonding, its vibrational frequencies are not expected to show much variation. It should be emphasized that the designations C~R,E, etc.. do not imply anything about the state of hydrogen bonding o f a residue, nor do they imply that a residue is necessarily part of a given structure. For example, whether or not an C~R residue will be in an a-helix depends upon the conformations o f its neighboring residues. When the conformations of a group of residues nearby in space are considered together, the participation o f given residues in certain topographical structures can be discerned. Three classes of topographical structures will now be defined. They are a-helices, extended structures, and unordered structures. E v e r y residue in a protein belongs to one of these three classes. A residue is said to be part of a right-handed a-helix if it is one of a set of at least three consecutive residues in the aR conformational state. This definition derives from the fact that three residues must be in the aR state in order that the CO group of the ith residue be hydrogen bonded to the N H group of the (i + 4)th residue. The regions assigned in Fig. 6 a r e more liberal in the assignment of values of 0h and tO than these definitions would imply. More correct values of these dihedral angles for some regular structures are given in Table 1II/~:~ Figure 7A illustrates a portion of a polypeptide chain showing the hydrogen bond that would exist between the CO group o f residue i and the N H group of r e s i d u e i + 4 when residues i + 1, i + 2, and i + 3 are all in the aa (or all in the aL) conformation. A residue that is in a right- or ~;:~I U P A C - - I U B C o m m i s s i o n on Biochemical Nomenclature, Biochemistry 9, 3471 (1970).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

/

I H N+

i

0 ~

105

\/.

'+

~Ci+l

C+

i H i

0 l,

, ~.Ni+2

H

0

/CI.

% / \c"~ \c " / ' J

i÷l

H

II

0

i+l I +

/ \

i÷2

H

0

i

l 0

C=O

•

N--H

Flo. 7. (A) A section of a polypeptide chain showing the hydrogen bond that would exist between the CO group of residue i and the N H group of residue i + 4 when residues i + 1.

i + 2, and i + 3 are all in the C~R(or all in the aL) conformation, (B) A schematic representation of the polypeptide chain shown in (A). The vertical lines represent the amide units and the N---C'~ and C~--C ' bonds of the backbone are represented together by the horizontal lines.

left-handed helix is designated aRH and o~Lu, respectively. The schematic diagram in Fig. 7B represents the polypeptide chain of Fig. 7A. The vertical lines are the amide units, the open circles representing the CO groups and the filled circles the N H groups. The amide groups are separated by the N - - C a and C~--C ' bonds which, together, are represented b y a single horizontal line. This simple schematic diagram will be very useful in later sections when examining the hydrogen-bonding patterns of longer runs of helix. It should be noted that, according to the definition given above for a helical topographical structure, isolated or runs of less than three aR residues are n o t part of o~-helices. Extended structures are defined as four or more consecutive residues with values of~,t~ in the E region. Sets of these extended chains which run in the same or opposite directions, forming interchain hydrogen bonds, are called parallel and antiparallei/3-sheet structures and are designated ~,, and ~AV, respectively. Extended conformations also appear that are not hydrogen bonding to other extended regions: these isolated structures are

106

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

simply designated by the symbol e, and their amide groups are assumed to be solvated. Those residues o f a protein that are not classified as part of c~-helical or extended structures are considered to be part o f the " u n o r d e r e d struct u r e " and are designated by the letter u. Hence, by definition, the u structure consists of residues with many different possible sequences of values of 05,0, which can lie in any of the five regions defined in Fig. 6. Therefore, a residue in a u structure does not have characteristic values of 05,0. Residues are classified as part of the u structure solely on the basis that they do not participate in a-helical or extended topographical structures. This point is emphasized here because the nature of the u structure must be understood to anticipate properly the manner in which its vibrational frequencies compare with those of c~-helical or extended topographical structures. In particular, since the u structure includes residues with any o f the five possible conformational states, it is incorrect to regard a u structure as having a characteristic set o f vibrational frequencies. Approximate values ~3 of the dihedral angles 05,t0, and ~o for residues in the regular topographical structures defined above are listed in Table llI. The definitions of these topographical structures were based solely upon the values of 05,to of sets of consecutive residues. As mentioned earlier, however, the vibrational frequencies of amide groups also depend on their state of hydrogen bonding. Every amide group is considered to be either hydrogen bonded to another amide group or solvated (by water). It turns out that, within any given type of topographical structure, different states of hydrogen bonding are possible for each amide unit. Hence, every residue in each topographical structure is further assigned to a topographical state in which both the values of 05,toa n d the state of hydrogen bonding of a residue are specified. It is these topographical states that might be expected to have distinct amide vibrational frequencies that could be used for frequency-conformation correlations. The different topographical states of residues in c~-helices will be considered first. The hydrogen-bonding pattern in helices varies with the length of the helical sequence. Figure 8 shows the hydrogen-bonding patterns for helical sequences o f n residues, f o r n = 3 to 6, using the schematic notation of Fig. 7B. For e v e r y n consecutive a states, it is necessary to consider the effects of the hydrogen bonding on n + 1 amide groups. For n = 3, it is seen from Fig. 8 that a single hydrogen bond between the CO group of amide group i and the N H group of amide i + 3 (residue i + 4) is formed. In general, when there are n consecutive helical residues, there are only n - 2 hydrogen bonds. For the four amide groups (i through i + 3) there are three different states of hydrogen bonding. For amide group i, the CO group is hydrogen-bonded: and for amide group i + 3, the N H

[5]

RAMAN A N D RESONANCE R A M A N SPECTROSCOPY I

107

1

ItII

n--3

aC+,-) a(-,-) a(-,-) a(-,+)

I n--4

I

IIIIt

1 l a(+,-) a(+,-) a(-,-) a(-,+) a(-,+)

t

,

I

ttttI

n=5

t I a(+,-) a(+,-) a(+,-) a(-,+) a(-,+) a(-,+)

f

n--6

"

I

f

IIttII '

I

'

I

a(+,-) a(+,-) a(+,7) a(+,+) cl(-,+) a(-,+) a(-,+) FIG. 8. Schematic representation of the hydrogen-bonding patterns in sequences of n consecutive helical residues when n = 3 to 6. See text for significance of the c~(+ or .+ or - ) designations.

group is hydrogen bonded, whereas amide groups i + 1 and i + 2 ave not hydrogen bonded (and hence, according to the model, are assumed to be solvated). To describe the state of hydrogen bonding of an amide group conveniently, the shorthand a ( + or - , + or - ) will be used where the first symbol between the parentheses indicates whether the CO group is either hydrogen bonded to the N H of another amide group (+) or is solvated ( - ) and the second symbol indicates whether the N H group is either hydrogen bonded to the CO of another amide group (+) ol-is solvated ( - ) . There ave four s t a t e s of hydrogen bonding possible: c ~ ( - , - ) , c~(- .+), c~( + , - ) , and

108

CONFORMATION;

OPTICAL SPECTROSCOPY

[5]

T A B L E IV DISTRIBUTION OF HYDROGEN-BONDED STATES IN HELICES OF DIFFERENT LENGTHS Number of consecutive helical residues (n) 3 4 5 6 7 8 9 10 11 12 13 14 15

Number of a(-,-) states

Number of a ( - , +) states

Number of a(+,-) states

Number of ~(+,+) states

2 1 0 0 0 0 0 0 0 0 0 0 0

I 2 3 3 3 3 3 3 3 3 3 3 3

1 2 3 3 3 3 3 3 3 3 3 3 3

0 0 0 1 2 3 4 5 6 7 8 9 10

a ( + , + ) . Using this shorthand for the case n = 3, the states of hydrogen bonding of the four amide groups of interest are designated (reading from left to right in Fig. 8) c d + , - ) , c ~ ( - , - ) , a ( - , - ) , and c ~ ( - , + ) . As the length of a helical sequence increases, the distribution of the four possible hydrogen-bonded states changes. These changes are illustrated in Fig. 8 for the cases n = 3 to 6 and summarized in Table IV for n = 3 to 15. The data in Table IV indicate that, in short (n < 15) sequences of helix, there are always significant amounts of three different types of helical topographical states. The lengths of helical sequences in globular proteins are generally not very large. In Fig. 9, a histogram illustrating the frequency of occurrence of helices of various lengths found in 15 proteins by X-ray crystallography is shown. '~4 It can be seen that there are few runs of helix longer than about 15; the average length is about 9-10 residues. Hence, within the short sections of helices found in proteins, the presence of three distinct topographical states has to be considered. To complete the description of helical topographical states, the subscripts RH and LH are added to the a( , ) shorthand to indicate whether the helix is right- or left-handed. Hence, there is a total of eight different types of helical topographical states: C~RH(--,--), aRr~(--,+), C~RH(+,--), ,~4The data plotted were taken from S. Tanaka and H. A. Scheraga, Macromolecules 9, 168 (1976).

[5]

109

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

8 oC~ "

[]

0

I 0

l

I

I

I0 Length of Helix(n)

I

I

20

FIG. 9. Histogram illustrating the frequency of occurrence of helices of various lengths found in 15 proteins by X-ray crystallography.

C~aH(+,+), ~L~(--,--), Cq,H(--,+), Cq,a(+,--), and O~LH(+,+)--all of which might be expected to have certain vibrational frequencies that are different by the criteria established earlier, since they all have different values of 6,+ and different states of hydrogen bonding. For example, in state ¢~(-,+), the N H group is hydrogen bonded. Referring to Table I1, this state might be expected to have a perturbed amide Ill frequency relative to that of the c ~ ( - , - ) state, since amide Ill modes involve substantial in-plane bending of the N - - H group. Similarly, the c~(+,-) state, in which the CO group is hydrogen bonded, might be expected to have a perturbed amide I frequency, since the amide 1 mode involves substantial C==O stretching. Likewise, the vibrational frequencies of the c~(+,+) state could differ still from the others. Finally, the vibrational frequencies of the (~RH(+,--) and OgLH("~-,--) states, the c~m~(+,+) and cq~H(+,+) states, etc., might be expected to differ since the values of 6,+ for right- and lefthanded helices are different. The different topographical states of residues in the three types of extended structures will be considered next. Parallel, ~p, and antiparallel, EAp, extended structures contain interchain hydrogen bonds between peptide amide groups, and every residue has either a CO or NH group (or both) that is hydrogen bonded. Residues in isolated extended structures (single chains), E, are not hydrogen bonded and, hence, are assumed to be solvated. Using the E(+ or - , + or - ) notation, as before, to specify the state of hydrogen bonding of the CO and N H groups of each amide unit, the seven possible topographical states of residues in extended structures are e ( - , - ) , CAp(--,+), EAp(-[-,--), EAp('4-,-{-), Ep(--,-~'-), Ep('4-,--), and E~,(+,+). A section of CAp structure consisting of three chains is shown in Fig. 10A (along with a schematic representation of it in Fig. 10B). It can be seen that residues in " i n t e r i o r " chains are EAp(q--,-}-) states and that

110

CONFORMATION : OPTICAL SPECTROSCOPY

a N-H------O=C

0=C N-H------O=C RHC\

N-H

/CHR

RHC\

C=O------H-N

H-N /CHR

RHC\

0=C \ N-H------ 0=C

/CHR

,N-H----- 0=C\ N-H

B

p(" .+)

F)c . 10 . (A) A section of an antiparallel0-sheet structure showing the hydrogen bonding between amide groups . (B) A schematic representation of the structure shown in (A) . See text for the significance of the E~v(+ or -, + or -) designations .

residues in "border" chains are either EAY(-,+) or EAY(+, - ) states . The relative amounts of these three states in an EAP structure will depend upon the number of chains in the sheet . For EAP structures with few chains (<5), as found in proteins,'" significant amounts of all three topographical states-EAI'( - ,+), EAP(+,-), and EAY(+,+)-will be present . The same arguments apply to El, structures . ''"J . S . Richardson, Proc . Nutt . Acad . Sci . U .S .A . 73, 2619 (1976) .

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

I 11

The assignment of topographical states to residues in u structures is not possible because of the variable nature of both their values of 6,O and their possible states of hydrogen bonding. Indeed, every residue in a u structure could be considered to be in a topographical state of its own. The term "'unordered topographical state" will be used in the following sections with the understanding that it refers collectively to all those residues whose topographical states cannot be formulated on the basis of the simple models for a and e structures discussed above.

Assignment of" Vibrational Frequencies to Specific Topographical States fi'om Studies ~( Model Compounds On the basis of the above discussion, there are eight helical, seven extended, and one (catchall) unordered topographical states whose presence in proteins may be detected, in principle, from their vibrational frequencies. The definitions of these 16 states have been developed in very general form. In practice, only a few of these states are accessible for study in model systems, and, hence, little can be said about most of them at this time. On the other hand, some of these are not expected to occur very frequently in proteins, and, hence, our lack of knowledge about them will be of little consequence. The existence of the more commonly occurring states, however, should be recognized, and efforts devoted to their future study, One must ultimately rely on R studies of model compounds to establish the frequencies of the topographical states defined above. Superficially, it would seem to be a straightforward task to obtain R spectra of synthetic homopolypeptides known to adopt c~-helical and extended structures, to correlate their observed frequencies with the topographical states defined above, and then to apply these correlations to R spectra of proteins. However, the application of such frequency-conformation correlations to the analysis of R spectra of proteins is not entirely proper, since the sections of c~-helix and extended structure that are found in proteins differ from those adopted by model polypeptides. As pointed out by Lord. "'~ synthetic homopolypeptides form long, regular sections of a-helix or extended structures in which the number and frequency of the R-active amide modes are formally restricted by the symmetry of the structure. The sections of c~-helix and extended structure that appear in globular proteins, on the other hand, are short, often distorted, and consist of a variety of possible side chains. As a result, the regular structures found in proteins are formally devoid of symmetry and there should be no sym';'; R. C. Lord. Int. Congr. Pure and Appl. Chem., 23rd, Vol. 7. p. 179. 1971.

112

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

metry restrictions on the activity of the various amide vibrational modes. Hence, the application to proteins of the frequency-conformation correlations formulated from studies of model compounds, while certainly of qualitative value, should not be expected to be entirely satisfactory. The conclusions that can be drawn about the vibrational frequencies of the amide I and llI and skeletal stretching modes of the 16 topographical states defined above from studies of model compounds will now be discussed. Helical topographical states will be considered first. Synthetic polypeptides of various amino acids known to form c~-helices have been studied extensively. :~In principle, the R spectra of helical synthetic homoor copolypeptides should depend on their degree of polymerization (DP) and on the heterogeneity of their chainlength. The average length of a helical sequence (L) in a homopolymer chain at a given temperature in the transition range depends on (among other factors) its DP--the larger the DP the larger the value of L. '~7 For very long chains (DP > 500), the average length of a helical sequence at the transition temperature is typically about 100. Since, according to Table III, it is L that determines the distribution of the topographical states present, the R spectra of synthetic homopolymers will depend upon their DP. Furthermore, the synthesis of a homopolypeptide produces a heterogeneous product (i.e., one with a distribution of chainlengths). When used without further fractionation, the spectra of such preparations will reflect the average properties of the chains of different DP present. Hence, in order to use synthetic homopolypeptides properly as models to evaluate the vibrational frequencies of various topographical states, the samples should be characterized with respect to both their DP and homogeneity of chainlength. Unfortunately, most of the R studies carried out on helical (and on other conformations of) synthetic polypeptides have not considered the possible effects of DP and heterogeneity of chainlength on the spectra. In general, however, the polymers studied were of sufficiently high DP that it can probably be assumed that L is large (and, hence, independent of DP) and that the R spectra reflect predominantly c~(+,+) states. The amide I band s of C~RH(+,+) states observed in the spectra of right-h anded c~-helical polymers are strong and lie close to the 1650-1660 cm -~ region. :~ In H.,O solutions, the amide I band overlaps with and is often obscured by the bending mode of H_,O which is centered ~'~ near 1645 cm ~. In D.,O solutions, however, the bending mode shifts ~'~ to about 1200 cm-; and the amide I' band of the O~RH(+,+) state is clearly resolved and lies in the 1635-1645 cm -~ region. The amide Ill bands of C~RH(+,+) states are weak and lie in the 1260-1290 cm ~ region. Upon deuteration, these bands shift ,;7 D. C. Poland and H. A. S c h e r a g a , " T h e r o y of Helix-Coil Transitions in Biopolymers.'" Academic Press, New York, 1970.

[5]

RAMAN

AND

113

R E S O N A N C E R A M A N SPECTROSCOPY

TABLE

V

CHARACTERISTIC RAMAN VIBRATIONAL FREQUENCIES {CM ~/ OF D I F F E R E N T TOPOGRAPHICAL STATES

Topographical

Amide

Amide

I"

liP'

stale

(~t~il( -- --

)

u(C'NC"Y'

h

h

~ RH[ --. + )

l,

p,

l,

~RII( + , -- )

h

l,

r,

~mj( + . + ) t5~l.ll(

__

.

__

]

1 6 5 0 - 1 6 6 0 (s) h

1260-1290 (w I h

(~1 H __ • __ )

h

h

~hH( + .

l,

I,

- )

ch H( + - + (~(

._ )

h

~ 1663 (s)

890-940 h l,

~,

--~'

- 9 0 0 Is)

h

t,

l,

~.xP( - - . + ) ~u'( + ,-- )

_/, t,

~, _j,

~, ~,

E w( + . + )

1 6 6 5 - 1 6 7 5 (s)

1235-1240

(s)

(s)

-- 1270 ( w ) ~,

890-940 (w)

%(- ,+)

_~,

%( + , - )

__~,

~,

~,

%( + . + )

__~,

~,

,,

u

--'

__'

__,

~,

" A b b r e v i a t i o n s : s. s t r o n g : w . weak. J' Not k n o w n . ' N o t u s e f u l - - s e e text for discussion,

to near the 950-1000 cm ' region (Table II). The v(NC"C') skeletal stretching m o d e s of a k , ( + , +) states are strong and lie in the 890-940 cm region.:~ There is a R spectrum available for a h o m o p o l y m e r of L amino acids that adopts a left-handed helix (poly-y-benzyl-L-aspartate), ';~ Its spectrum can be used similarly to assign the vibrational frequencies of the aLrt(+, +) state. The amide I band is strong and lies at 1663 cm-~, and the strong , ( N C ~ C ') skeletal stretching m o d e s lie at about 900 cm -~, The amide 111 vibration has not yet been assigned. T h e s e assignments are summarized in Table V. There are presently no experimental data available from which the vibrational frequencies of the c~(-,-), a ( - , + ) , or ~ ( + , - ) states of either right- or left-handed helices can be assigned. Since no left-handed helices have been found in globular proteins, our ignorance of the frequencies of a l , H ( - , - ) , a L , ( - - , + ) , and aLH(+,--) states is of no practical importance. "~ B . G . F r u s h o u r

and J. L. Koenig,

Biopolymers,

14, 2 1 1 5 11975).

114

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

The vibrational flequencies of the O~RH(-- ,-- ), C~tt(--,+), and c ~ H ( + , - ) states, which are found in the short helical runs of proteins, are important and should neither be neglected nor assumed without p r o o f to be identical with those of the c ~ ( + , + ) state. The study of synthetic polypeptides that adopt extended structures has been limited to the ~.,,, structure. :~ Most o f the E.w structures examined have been solids or gels, since the formation of eAj, structures in solution leads to aggregation and precipitation. It can, therefore, be assumed that the E.~j, structures studied consisted of large numbers of antiparal]el chains, side by side, and that appreciable amounts of only the EAe(+,+) state were present. In the spectra of these polymers, the amide I band is strong and is found close to the 1665-1675 cm ' region. :~ A strong amide lIl band is usually present in the 1235-1240 c m - ' region ;~ and a weaker c o m p o n e n t m a y a p p e a r :~near 1270 cm- '. The u(NC"C') skeletal stretching modes of the EA~,(+,+) state have not been observed, p r e s u m a b l y b e c a u s e they are weak. There have been no experimental studies from which data for the EAj,(--,+) and eAj,(+,-) states can be derived. H o w e v e r , since these states o c c u r in proteins, their presence should not be ignored, nor should they be assumed, a priori, to have the same vibration frequencies as the EA~,(+,+) state. Finally, the vibrational frequencies of e ( - , - ) , E~,(-,+), E~,(+,-), and ~,(+,+) states cannot, at present, be assigned either since suitable models are not available for study. The assignment of characteristic vibrational fi'equencies to the unordered state of proteins is clearly not possible since, by definition, this category includes a large number of different conformations. In spite of this, in the recent literature, characteristic fi-equencies for the u state of proteins have been proposed by observing the R scattering from charged, randomly coiled polypeptides. This procedure is incorrect for two reasons. First, even if the u state could be considered to have characteristic vibrational frequencies, unfolded, randomly coiled polypeptides cannot be used as models for the u state of a folded protein. The residues in the coil state of a random polypeptide chain m a y take on an ensemble of different conformations. In a native protein, however, every residue that is not part of a regular c~ or e structure (and which is then classified as a u state in the present terminology) adopts a specific conformation. Hence, the coil state of an unfolded polypeptide chain is not the same as the u state of a residue in a globular protein.":' Therefore, randomly coiled polypeptides cannot be used as models for the unordered state in globular proteins. The indiscriminate use of the term "coil s t a t e " to describe the state of residues in both folded and unfolded polypeptide chains has led to this confusion. '"'C. B. Anfinsen and H. A. Scheraga, Adv. Protein Chem. 29, 205 (1975).

[.5]

R A M A N A N D R E S O N A N C E R A M A N SPECTROSCOPY

115

Second, as mentioned earlier, the u state of either a folded or unfolded polypeptide chain consists of m a n y different conformations: therefore, it is unlikely to have unique values for its vibrational frequencies. The R scattering o b s e r v e d from the u structure will clearly be due to a weighted average over all the different conformational states in the chain. The positions of the frequency m a x i m a of the resultant bands (which are broad) are not indicative of any single characteristic conformational state. Finally, to say that a protein is 60% unordered does not provide much information about the actual conformation of the residues involved. For all the above reasons, the c o m m o n l y assigned values of the vibrational frequencies of the unordered structure are not useful for detecting the conformational states of proteins. It should be pointed out that, while the discussion here has centered around the different R frequencies of topographical states, the R intensities of amide group modes are also sensitive to conformation. For example, it has been mentioned a b o v e that the amide IIl band of the e w( + ,+t state is strong, but that the amide lll band of the C~RH(+,+) state is weak. When poly-L-lysine undergoes the ~ R H ( - { - , " ~ ) ~ EAI,(@,-t-) transition, it is a c c o m p a n i e d by a net increase in the amide I11 band intensity. TMThe net decrease or increase in the intensities of R bands brought about by changes in conformation or environment is referred to as Raman hypochromism or Raman hyperchromism, respectively. These terms derive from the analogous h y p o c h r o m i c or h y p e r c h r o m i c effects found for the intensities of absorption bands in the ultraviolet spectra of biopolymers. In fact, the correspondence of the direction o f the changes in intensity observed in ultraviolet and R spectra implies a direct correlation between the two phenomena. It can be seen easily from Eq. (15) that the intensity of a R band is related by means of a sum-over-electronic states to the intensities of various ultraviolet bands through the electronic dipole transition m o m e n t , (M)r~. Using Eq. (15), a direct relationship between R and ultraviolet hypo- and hyperchromic effects has been verified recently. TM In s u m m a r y , the R technique can be used to detect the presence of ~¢H(+,+) and e w ( + , + ) states in globular proteins. The presence of ~m~(+,+) states is characterized by the amide I intensity near 1655 cm strong u(NC~C ') skeletal stretching bands in the 900-950 cm -~ region and the absence of spectral intensity in the 1235-1240 c m I region. The presence of EAp(+,+) states can be deduced most reliably from their strong amide ill bands near 1235-1240 cm ~. The amide i intensity near 1670 cm ' is also diagnostic of the CAp(+,+) state. It should be noted that the shapes of amide 1 and ill bands of R spectra :"T. J. Yu, J. L. Lippert, and W. L. Peticolas. Biopolymers 12, 2161 11973). r' P. C. Painter and J. L, Koenig, Biopolymers 15, 241 (19761.

116

CONFORMATION"

OPTICAL SPECTROSCOPY

[5]

of proteins are influenced by contributions from glutamine and asparagine side-chain amide modes as well as from other side-chain modes, such as tyrosine ring modes. In addition, proline modes contribute to the intensity in the amide I and III regions. Since asparagine, glutamine, and proline together comprise about 12% of all amino acids in globular proteins, their contribution to the amide I and III contours of the R spectra of proteins, taken together with that of all the other side chains, is not negligible: hence, the prospects of using these band contours for quantitative estimation of the presence of backbone topographical states do not appear to be encouraging. The contribution of the nonamide side-chain modes to the amide III contour can be determined by examining the R spectrum of the protein when all of its amide groups have been deuterated. The intensity that remains in the amide III region after deuteration can be attributed to nonamide vibrational modes.

Conformationally and Environmentally Sensitive Side-Chain Vibrational Modes Many of the bands observed in the R spectra of proteins can be attributed to the side chains. For example, the COO- symmetrical stretch, the CH2 bending mode, the intense aromatic ring modes of phenylalanine, tyrosine, and tryptophan, the S--S and C--S stretches of cystine, the C--S stretches of methionine, and many others have been detected. 7~ While, in principle, the frequencies of any of these bands could be used to provide information about side-chain conformation or environment, only a few studies of relevant model compounds have been carried out and, hence, only a few correlations now exist. The most extensively studied side-chain vibrational modes have been the S--S stretching modes of cystine residues, which are strong and lie in the 480-540 cm -~ region. 7:~-~:~From an analysis of the R spectra of model disulfides, the S-S stretching frequency has been shown to be sensitive to ~ R. C. Lord and N. T. Yu, J. Mol. Biol. 50, 509 (1970). 7:~H. Sugeta, A. Go, and T. Miyazawa, Chem. Lett.. p. 83 (1972). ~ H. Sugeta, A. Go, and T, Miyazawa, Bull. Chem. Soc. Jpn. 46, 3407 (1973). 7:'E. J. Bastian, Jr., and R. B. Martin, J. Phys. Chem. 77, 1129 (1973). ;'~ H. E. Van Wart, A. Lewis, H. A. Scheraga, and F. D. Saeva, Ptvc'. Natl. Acad. Sci. U.S.A. 70, 2619 (1973). r; R. B. Martin, J. Phys. Chem. 78, 855 (1974). 7~ H. Sugeta, Spectrochim. Acta 31A, 1729 (1975). 7, H. E. Van Wart. F. Cardinaux, and H. A. Scheraga, J. Phys. Chem. 80, 625 (1976), ~" H. E. Van Wart and H. A. Scheraga, J. Phys. Chem. 80, 1812 (1976). ~ H. E. Van Wart and H. A. Scheraga, J. Phys. Chem. 80, 1823 (1976). ~eH. E. Van Wart, H. A. Scheraga, and R. B. Martin, J. Phys. Chem. 80, 1832 (1976). ~:~H. E. Van Wart and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 74, 13 (1977).

[5]

R A M A N A N D RESONANCE RAMAN SPECTROSCOPY

117

TABLE Vl SUMMARY OF THE CONCLUSIONS THAT CAN BE DRAWN ABOUT THE VALUES Or: x(SS-CC) AND x(CS-SC) OF PRIMARY DISULFIDES FROM THE OBSERVED VALUES OF v(S-S)"

Observed value of ~,(S-S) (cm- 1)

Ix(SS-CC)I" (degrees)

'i x(CS-SC),

480-50~_

--'

{ 115-181/0-65 (use Fig. 11)

510 _+ 5

{50-180 / 50-180J

85 _ 21)

525 _+ 5

0-50 / 50-180J

85 + 20 -

540+5

(degrees)

{0-50}

-

85 _+ 20

0-50

" From H. E. Van Wart and H. A. Scheraga, J. Phys. Chem. 80, 1823 (1976). ~' One range of dihedral angles is specified for each C - - S bond of the CCSSCC moiety. ' No information is obtainable.

the X~ and X:~ side-chain dihederal angles (i.e., the SS-CC and CS-SC dihedral angles, respectively) of cystine. Hence, from the value observed for the S-S stretching frequency, certain conclusions about the conformation of cystine side chains in proteins can be drawn. A summary s~ of the information provided is given in Table VI and Fig. II. I

i

I

I

520

( ~ F O -

-

I E o 09 L (,9

500

48C

;

I

I 40 IX (CS-SC)

1

I 80

I (degrees)

FIG. I I. Relationship between the S - S stretching frequency, t,(S-S), and the C S - S C dihedral angle, I x(CS-SC) I , for primary disulfides. Adapted from H. E. Van Wart and H. A. Scheraga, ./. Phys. Chem. 80, 1823 (1976).

1 18

CONFORMATION;

OPTICAL SPECTROSCOPY

[5]

R spectra of proteins often show a pronounced doublet with peaks near 850 and 830 cm -~, respectively, attributable to tyrosine. In an extensive study of model compounds for tyrosine residues in proteins, it has been established ~4 that this doublet is due to a mechanical resonance interaction (referred to as Fermi resonance 45) between the ring-breathing vibration and the overtone of an out-of-plane ring-bending vibration of the tyrosine side chain. As a result, the intensity ratio of the two components in the doublet depends on the relative frequencies of these two vibrations. Since these frequencies, in turn, are sensitive to the state of hydrogen bonding and ionization of the phenolic hydroxyl group, the relative intensities of the bands for this doublet in proteins can be correlated with the state of the phenolic hydroxyl group in tyrosine residues. Three states of tyrosine side chains may be distinghished from the value of the I(850) : I(830) ratio--those that are " n o r m a l " or exposed on the surface of the protein, those that are buried (and considered to be strongly hydrogen bonded), and those that are ionized; these three types are designated by the letters N, H, and I, respectively. The values o f I(850):I(830) are near 10:8 for N states, in the 10:7 to 3:10 range for H states, and near 7 : 10 for I states. When the pH is less than about 10, all the tyrosines are protonated and only N and H states need to be considered. For a protein with more than one tyrosine, the value of the I(850) : I(830) ratio can be used to estimate the number that are in the N and H states. In Table VII, the results obtained when these correlations are applied to several proteins are listed. These results have been found to be in good agreement with conclusions reached by other m e t h o d s ? ~ The intensities of any of the aromatic ring modes of tyrosine, phenylalanine, and tryptophan have the potential of providing information about their environment through R hypo- or hyperchromic effects, in a manner similar to that observed for the amide III band. In fact, the ultraviolet spectra of tyrosine residues in proteins are known to be sensitive to their environment. ~ Furthermore, the high intensity of aromatic ring modes may arise from a small degree of preresonance enhancement from their ultraviolet absorptions. If this is so, their R intensities would be expected to be sensitive to changes in their ultraviolet absorptions. R intensity changes in the 644 cm -~ band of tyrosine residues in ribonuclease ~; and cobramine B ~7 have been reported and rationalized in terms of the environment of the tyrosines. ~4 M. N. Siamwiza. R. C. Lord, M. C. Chen, T. Takamatsu, 1. Harada, H. Matsuura, and T. Shimanouchi, Biochemistry 14, 4870 (1975). ~:' H. A. Scheraga, "'Protein Structure." Academic Press, New York, 1961. ~;N. T. Yu, B. H. Jo, and C. S. Liu, J. Am. Chem. Soc. 94, 7572 (1972). S;N. T. Yu, B. H. Jo, and D. C. O'Shea, Arch. Biochem. Biophys. 156, 71 (1973).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

[ 19

TABLE VII CLASSIFICATION OF TYROSYL RESIDUES IN CERTAIN PROTEINSII

Protein (No. of tyrosyl residues~ Lysozyme (3) Ribonuclease A (6}

Insulin 14)

Erabutoxin A (11 Cobramine B (3)

State of sample

Observed doublet ratio 1(8501 : 1~8301

Classification of tyrosyl residues ~'

Crystals Aq, pH 4.5 Powder Aq, pH 5.0and 8.9 Aq, pH 1.7 Crystals Aq, p H > 13 Aq, pH 2.4 and 8.3 Aq, pH 7 Powder Aq. pH 7, 30°C Aq, 85°C

10:9' 10: 10' t(1:9'~ 8:10 '~ 1(1: 1(1'; 10: 8' g: 10" 10:8" 5: 10' 5 : 10" 6: 1(1'; 10:8"

3 Nov 2 N + I H 2N + 1H 4 N + 2 H or 5 N + I H 3N + 3H 4 N + 2H 4N 41or31+ IN 4N I H 3H 3 H or 2 H + I N 3N

" Adapted from M. N. Siamwiza, R. C. Lord, M. C. Chen, T. Takamatsu, 1. Harada, H. Matsuura, and T. Shimanouchi, Biochemisoy 14, 4870 (19751. *' Symbols designate: N, normal: H, hydrogen-bonded, and 1, ionized. " N. T. Yu and B. H. Jo, Arch. Biochem. Biophys. 156,469 (1973}. " N. T. Yu, B. H. Jo, and C. S. Liu, J. Am. Chem. Soc. 94, 7572 (19721. N. T. Yu, C. S. Liu, and D. C. O'Shea, J. Mol. Biol. 70, 117 (19721. 1. Harada, U.S.-Japan Joint Seminar, The Raman Spectroscopy of Biologic~d Molecules, Cleveland, Ohio, 1974, p. 15. N. T. Yu, B. H. Jo. and D. C. O'Shea, Arch. Biochern. Biophys. 156, 71 (1973).

Examples o f Spectral Analysis and Interpretation o f Nonresonance Raman Protein Spectra Several representative R spectra of proteins from studies that have a p p e a r e d in t h e r e c e n t l i t e r a t u r e will n o w b e c o n s i d e r e d t o i l l u s t r a t e t h e a p p l i c a t i o n s o f t h e c o r r e l a t i o n s d i s c u s s e d in t h e p r e c e d i n g s e c t i o n s . F i g u r e 12 s h o w s t h e R s p e c t r a o f a l y o p h i l i z e d p o w d e r a n d o f a n a q u e o u s s o l u t i o n o f b o v i n e p a n c r e a t i c r i b o n u c l e a s e A o b t a i n e d b y Y u et a/. sr F o c u s i n g a t t e n t i o n o n t h e a q u e o u s s p e c t r u m , t h e b a c k b o n e vib r a t i o n a l f r e q u e n c i e s will b e c o n s i d e r e d first. In t h e a m i d e I11 r e g i o n , b a n d s a r e o b s e r v e d a t 1239 a n d 1265 c m '. T h e b a n d a t 1239 c m ~ i n d i c a t e s t h e p r e s e n c e o f t h e e a ~ ' ( + , + ) s t a t e , w h i l e t h e b a n d a t 1265 c m ' s u g g e s t s t h e p r e s e n c e o f t h e C~RH(+,+) s t a t e . T h e X - r a y s t r u c t u r e o f r i b o n u c l e a s e A c o n f i r m s t h e p r e s e n c e o f t h e s e t w o s t a t e s in t h e c r y s t a l l i n e phase, Recently, Painter and Koenig ~ have shown that some of the inten~ p. C. Painter and J. L. Koenig, unpublished data, cited by Frushom- and Koenig. :=

120

CONFORMATION:

OPTICAL

SPECTROSCOPY

Arnide I

[5]

(A) Lyophilized Powder

Amide Tl'r

C'=-C_N Tyr

I IO

/

~

~nPhe

/~)r-- O

c

l

c:

~

_

J

~

_

1600

t

_

_

1400

_

(B] Aqueous Solution

II

_

t

1200

_

_

1000

pH B.89

_

J

_

800

_

~

600

Av(em "l ) FIG. 12. Raman spectra of ribonuclease A in the solid and aqueous solution. (A) Spectrum of the lyophilized powder in 0% relative humidity: slit width, 200 tzm (4 cm '); sensitivity, 5000 counts per second full scale; rate of scan, 10 cm-'/min; laser power at the sample, 153 mW; laser line, 514.5 nm. (B) Spectrum of the aqueous solution (200 mg/ml) at pH 8.89: slit width, 200 ~m (4 cm-t); 5000 counts per second full scale; rate of scan, 25 cm-'/min; laser power at the sample, 200 roW; laser line, 514,5 nm. Adapted from N. T. Yu, B. H. Jo, and C. S. Liu, J. Am. Chem. Soc. 94, 7572 (1972).

sity of the 1265 c m -~ b a n d r e m a i n s after d e u t e r a t i o n . H e n c e , this b a n d c a n n o t be d u e e n t i r e l y to an a R ~ ( + , + ) a m i d e III mode. It was suggested that the r e m a i n i n g b a n d was d u e to a t y r o s i n e m o d e . H e n c e , the b a n d at 1265 cm - ' is p r o b a b l y d u e both to the ~RH(+,÷) state a n d a t y r o s i n e side-chain mode. T h e a m i d e I b a n d is u n r e s o l v e d a n d lies at 1667 c m - ' . This f r e q u e n c y is, in itself, i n c o n c l u s i v e in suggesting the p r e s e n c e of p a r t i c u l a r topographical states, b u t is c o n s i s t e n t with a s u p e r p o s i t i o n of b a n d s d u e to

[5]

RAMAN A N D RESONANCE RAMAN SPECTROSCOPY

121

- a l l ( + , + ) and cAp(+,+) states, which would be expected to contribute intensity to the amide 1 contour near 1655 and 1670 cm ~, respectively. The strong skeletal stretching modes in the 890-940 cm ' region are also indicative of the c~au(+,+) state. Turning to the side-chain modes of the aqueous spectrum, the tyrosine doublet at 854 and 834 cm ~ has an intensity ratio, 1(854): I(834), of 8: 10. This implies that, of the six tyrosine residues in the protein, half (three) are exposed to the solvent and the other half (three) are strongly hydrogen bonded in the interior (see Table VII). The S - S stretching frequency of 516 cm ~ indicates that the cystine residues have no strained disulfide bonds and no values of the S S - C C dihedral angle below about 50°. From a comparison of the spectra of the aqueous and lyophilized powder samples shown in Fig. 12, it can be seen that the intensities of the tyrosine ring vibrations at 644, 832, and 852 cm ' are sensitive to the change in state. The increase in the intensity of the 644 cm ' band and 1(854): 1(834) ratio to 10:9 can be interpreted as being due to a change in the local environment of one or two of the buried, hydrogen-bonded tyrosine residues. The narrower bandwidth of the S-S stretching band in the aqueous compared to the powder spectrum is probably due to a greater uniformity in the conformations o f the cystine residues in solution. The backbone vibrational frequencies do not change appreciably on going from the aqueous to powder phases, indicating that no backbone conformational changes have occurred. A listing of the bands that appear in the R spectrum of ribonuclease A at pH 5 (the spectra at pH 5 and and pH 8.89 are very similar) along with their assignments, as given by Chen and Lord, ~' is shown in Table VIII. The changes in the R spectrum of ribonuclease A brought about by temperature variation have been used by Chen and Lord ~' to study its thermal unfolding. These authors used changes in many of the spectral features shown in Fig. 12 due to backbone and side-chain modes to follow the changes in conformation in different regions of the protein. Insofar as some of these observed spectral changes (such as the band half-width of the four disulfide stretching frequencies) could not be correlated with s p e c i f i c conformational changes in the protein, they represent the phenomenological use of the R technique to study the course of protein unfolding. These spectral changes are plotted in Fig. 13, and have been used to help establish a stepwise unfolding process tk~r the protein. As another example of a nonresonance R spectrum, we consider porcine insulin. The spectra of powdered insulin and an insulin single crystal, obtained by Yu e t al., ''~' are shown in Fig. 14. Differences in ~" M. C. Chen and R. C. Lord, Biochemisto' 15, 1889 (1976). .... N. T. Yu. B. H. Jo, R. C. C. Chang, and J. D. Huber, Arch. Biochem. Bk~phys. 1611, 614 (1974).

TABLE VIII RAMAN FREQUENCIES AND INTENSITIES OF RIBONUCLEASE A IN AQUEOUS SOLUTION"

Frequency/' (cm- ') 412 (0) 440 (0) 496 (0 s) 516 (3l 555 (0) 594 (0) 605 (0l 622 (1) 644 (3) 657 (4) 675 (2 s i | 7/4 (0 s ) | 724 (2) J 755 (1) 808 (I) 834 (5) / 854 (4) J 892 (3) "~ 902 (3l | 918 (0 s ) | 937 ( 4 ) ) 960 (0) 982 (9) 1003 ( IOI 11)15 (0 s) 1030 (3} 1062 (4) } 1082 (d) 1106 (4) 1125 (3) 1154 (I) 1180 (2) 1191 [0 s/ 1210 (31 1239 1111 1263 (10) 1284 (0 s) 1315 (8) I 1324 (1 s ) | 1337 (3) J 1399 (4) 1412 (51 1420 15l 1447 (lO) 1585 (0 s) 1603 (I s) 16~9 (3) 1668 (24)

Tentative assignment"

v(S-S)

Phe Tyr

v(C-S)

Tyr

/~(Ca-C )

SO42 Phe Phe

v(C"-NI

Tyr and Phe Tyr and Phe Amide Ill Amide I11 and Tyr Amide Ill

y(CH.,)(?) vICOz ) ~5(CH,,)

Tyr and Phe Amide I and H._,O

" Adapted from M. C. Chen and R. C. Lord, Biochemistry 15, 1889 (1976). /, Numerical figures in parentheses are relative peak intensities with that at 1447 c m - ' taken as 10. The letter s denotes a shoulder. ' v means stretching. ,5 means deformation, and y means twisting.

[5]

123

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

I 5101

l --I

I

I

I 62.0°C

-- I ~

B3Ocm-f\ ~ \ n f t "

1 41)

+"

c

t

I

4--T~,+

1

600

3.0

L~/~:~" 63.0 ; 40

854 cm-I

2.0

~ k

u (C-Sl I

30 11.0

I

I

I

q t

I

I

I

Jt~('-"

1250 cm "I (Amide in')

.1= 2O

/

3 I

60.0"C/

?

~oo

I

15

,/ 9.0

__~/ I

I

t

t

.--~,~...~

'E

937 cm-t 3.0 v(Ca-C)

515 ~

~ , Lt '35°c lID

g "

\,. ?0 30

4o

I

50

I

60

l

70

505

u (S-S) l

8o

30 T e m p e r a t u r e (°C)

40

l

50

l

60

l

70

FIG. 13. Thermal transition curves of ribonuclease A as monitored by the intensity tyrosine doublet at 830 and 854 cm ~, the intensity of the amide II1 band at 1250 cm intensity of the v(C~-C) band at 937 c m - ' , the intensity of the v(C-S) band at 657 cm the frequency and half-width of the u(S-S) band. Adapted from M. C. C h e n and R. C. Biochemist~ 15, 1889 (1976).

80 of the ~, the ~. and Lord,

conformation between insulin molecules in the two physical states are reflected by the regions of the spectrum indicated by arrows--namely, the amide I, amide III, u(NC~C'), v(C--S), and u(S--S) regions. The spectrum of deuterated insulin (which was useful for making band assignment, :'° particularly in the amide Ill region) was also obtained by these workers. The spectra of the insulin (a) and deuterated insulin (b) crystal in the amide Ill region are shown in Fig. 15 along with the difference spectrum ( a - b) between the two forms. The difference spectrum should represent only those bands due to amide Ill modes (not those due to tyrosine side chains, etc., which do not shift on deuteration), The amide 1 band at 1658 cm -~ in the R spectrum of the insulin single

124

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

Amide Tn"

,,::r

t

~

o

Amide I

(A] Single Crystal

m Nou ~_ - -

=~ f,-

~o

Mother Liquor

(B) Air Dried

II II

1600

1200

Powder

800 /~v

[

/

/

400

(cm "1)

FIG. 14. Raman spectra of porcine insulin single crystal and air-dried powder. (A) Spectrum of the single crystal in equilibrium with its mother liquor: slit width, 4 cm-~; sensitivity, 2500 counts per second full scale: rate of scan, 10 cm Vmin: time constant, 6 sec; laser power at the sample, 80 mW: laser line, 514.5 nm. (B) Spectrum of the air-dried powder: slit width, 4 cm ~: sensitivity, 5000 counts per second full scale: rate of scan, I0 cm '/mini time constant, 3 sec: power at the sample, 80 roW; laser line 514.5 nm. Adapted from N. T. Yu, B. H, Jo, R. C. C. Chang, and J. D. Huber, Arch. Biochem. Biophys. 160,614 (1974).

crystal shown in Fig. 14 is probably due predominantly to the aRH(+,+) state. The shoulder at 1681 cm -~ might be due to a state not encountered in model studies, since no amide I bands with frequencies this high have been encountered. The skeletal stretching m o d e s in the 940 c m - ' region are also indicative of the c~a,(+,+) state. Amide II1 components at 1240, 1269, 1284, and 1303 c m -1 are apparent f r o m Fig. 15. The unambiguous assignment of all these bands to given states on the basis of the information presented in Table V is not possible. The band at 1240 cm ~ is probably due to the e A A + , + ) state while either of the bands at 1269 and 1284 c m - '

[5]

R A M A N A N D R E S O N A N C E R A M A N SPECTROSCOPY

125

0

lP,

C

t

I

l

I

13O0 1200 L~v (crn -I)

i

FIG. 15. R a m a n s p e c t r u m of insulin (a) and deuterated insulin (b) single crystal in the amide l[l region and the difference (a - b) between these spectra. The instrumental conditions were similar to those for the s p e c t r u m in Fig. 14A. Adapted from N. T. Yu, B. H. Jo, R. C. C. Chang, and J. D. Huber, Arch. Biochem. Bit)phys. 160, 614 (1974).

may be due to the c~aH(+,+) state. The band at 1303 cm-' cannot be assigned on the basis of what is presently known from model studies. A listing of the bands in the R spectrum of bovine insulin crystals together with band assignments, as given by Yu et al.," is shown in Table IX. The degree of uncertainty of the above band assignments is large and reflects the early stage in the development of backbone frequencyconformation correlations that exist at the present time. It is clear that the "three-state'" model for interpreting protein R spectra in terms of backbone conformation, whereby all residues in proteins are classified as part of either a, /3, or u structures, is grossly oversimplified and inadequate. This can be seen both from the theoretical considerations of an earlier section and the problems of interpretation exemplified by the amide Ill contour of Fig. 15. By using the model for topographical states ~" N. T. Yu. C. S. Liu, and D. C. O ' S h e a , J. Mol. Biol. 7il, 117 (19721.

TABLE IX RAMAN FREQUENCIES AND INTENSITIES OF BOVINE INSULIN CRYSTALS" Frequencies I' (cm-~) 333 (0.9) / 410 (0-8) | 467 (0-8) I 495 (1.2) "~ 515 (3.2) / 563 (1- 0) 624 (2.0) 644 (3' 6) 668 (2' 0) "~ 678 (1.0s)J 725 (0"8) } 747 (0.8) 770 (0.8) 814 (I "4s) 832 (4.4) "~ 854 ( 5 " 5 ) ) 900 (2.0) 934 (2.0s) ~, 946 (3.2) | 963 (2.9) J 1004 (10-0)/ 1032 ( 3 . 3 ) ) 1112 (1.5s)'] 1128 (1"8) | 1162 (0-9) J 1177 (2 '4) 1212 (4.6) 1239 (5.0s)/ 1270 (5.3) | 1288 (4.7s)J 1322 (2.0s)1 1344 (4.0) } 1367 (I .6s)j 1425 (2.5s) 1450 (5.0) "~ 1462 (4.6s)J 1587 (1- 3) 1607 (3.6) 1615 (3.6) 1662 (4.6) "~, 1685 (4.0s)J

Tentative assignment'

Skeletal bending /~(S--S)

Phe Tyr v(C--S) of Cys Skeletal bending

Tyr v(C--C) Phe v(C--N) Tyr Tyr and Phe Amide Iii

CH deformation Symmetrical CO._, stretching CH2 deformation Phe Phe and Tyr Tyr Amide I

" Adapted from N. T. Yu, C. S. Liu, and D. C. O'Shea, J. Mol. Biol. 70, 117(1972). b Numerical figures in parentheses are relative peak intensities with that at 1004 cm-' taken as 10. The letter s denotes a shoulder. " v means stretching vibration,

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

127

developed in an earlier section, many of the theoretical deficiencies of the three-state model can be remedied. H o w e v e r , systematic and innovative studies will be required to characterize further the vibrational frequencies of the various topographical states that have been defined in this model. It seems likely that, when such studies have been completed, the origins of the amide llI bands in Fig. 15 will be better understood. Referring to the side-chain R bands that can be seen in Fig. 14, the tyrosine doublet at 854 and 829 cm -~ has an intensity ratio I(854) : I(829) close to 10:8 indicating that all four tyrosine residues are normal and exposed to the solvent. This conclusion disagrees with those drawn from other types of studies, in which one or two of the tyrosines have been found to be ~ a b n o r m a r ' (see Table V of Siamwiza et al.~4). The S-S stretching band at 492 cm-~ indicates the presence of a strained S-S bond estimate& ~ from Fig. 11 to have a C S - S C dihederal angle of about 35 °. This probably arises from the small loop in the A chain due to the distllfide bond between cystine residues 6 and 11, and it seems reasonable to conclude that the 492 c m - ' band is due to this cystine residue. The S - S stretching band at 514 cm 1 is due to an unstrained cystine residue(s) with S S - C C dihedral angles above 50 °. The band at 538 cm -~, if due to an S-S stretching mode, would indicate the presence of a cystine residue with low t0-50 °) values ~ of the S S - C C dihedral angle about both of its C - - S bonds. Resonance Raman Spectra of Proteins The types of vibrational modes observed in rR spectra depend upon the chromophore, and, hence, a general review of commonly occurring rR bands is not possible. H o w e v e r , the great majority of recent biological rR studies can be grouped, according to the chromophore, into one of several classes, each of which produces characteristic spectra. The commonly studied groups of compounds that are inherently chromophoric in the visible include heme proteins, copper and nonheme iron metalloproreins, carotenoids and visual pigments, chlorophylls and vitamin B~ derivatives. The studies of rR labels bound to proteins thus far has been limited to chromophoric enzyme substrates or inhibitors and azo dyes. In the following sections, the interpretation o f the rR spectra of representative examples of a few of the above classes is considered. H e m e Proteins

The heme group of heine proteins has been the single most important and most frequently studied class of chromophores. Its rR spectra have

128

CONFORMATION" OPTICAL SPECTROSCOPY CH 3

H"C

R

\C

C/

/

\

C%N C--C

HOOC- CHz-C

I ]

H/

\

..

C "---~ C \R

I __c/N ~ c

c

[5]

II - C\H

/

Ci ~ C CIH2 CHz

CH 3

I

COOH HEMOGLOBIN

R=-CH=CH 2

CYTOCHROME C R = - - C--CHCH 3 I

S I

CYSTEINYL

FIG. 16. Structure of heme, indicating pyrrole substituents which occur in hemoglobin and cytochrome c. Adapted from T. G. Spiro and T. C. Strekas, J. A m . Chem. Soc. 96, 338 (1974).

provided both the stimulus for extensive, in-depth experimental studies and for examination of the predictions o f quantum theories of R intensities. The rRS of these c o m p o u n d s have been reviewed in detail elsewhere. ~':~'' In this section, w e summarize the key characteristics of heme rR spectra. These proteins illustrate many o f the different phenomena and potential pitfalls associated with the interpretation of rR spectra. The structure of the heme group is shown in Fig. 16. The electronic spectra of all heine proteins consist of an intense (e - 10:' M-~ cm-~) Soret (or y) band near 400 nm and a pair of w e a k e r (e -- 104 M -~ cm -~) bands, called the a and 13 (or Qo-o and Q0 1) bands, near the 500-600 nm region? These features are illustrated in Fig. 17 for ferrocytochrome c. In addition, there can be weaker bands in the visible region that are due to charge transfer transitions from the porphyrin to the metal. In some cases, mixing between a, /3, or Soret and porphyrin-metal charge transfer transitions can occur, resulting in rather complex absorption spectra. Using the D4h point group for the heme molecule, molecular orbital "~T. G. Spiro, Ace. Chem. Res. 7, 339 (1974).

[5]

129

R A M A N A N D RESONANCE RAMAN SPECTROSCOPY

Visible(~8)Resononce~ - 30

150 ?

o ~Et0c

A

i

=<

~

.

7

~_ Soret

20 _O ,c

~J

IResonance'

o t

50 5O

4O0

450

5OO

I 550

O 6O0

k (nm)

FIG. 17. The near ultraviolet (Soret) and visible (c~/3) absorption spectrum of ferrocytochrome c. The arrows span the regions in which resonance with each of the two kinds of optical transitions dominates. Adapted from T. G. Spiro and T. C. Strekas, J. Am. Chem. Soc. 96, 338 (1974).

studies :':~predict that the highest filled orbitals are ofalu and a2~, symmetry and that the lowest empty orbital is ofeg symmetry. The c~ band is due to the (12u(Tr)---~eg('n'*) electronic transition and the Soret band to the a ldTr) --~ e~(~-*) transition. Both transitions are of E, symmetry, polarized in the plane of the heme, and are allowed. Configuration interaction between these nearly degenerate transitions leads to addition and cancelation of the transition dipoles resulting in the intense Soret and weaker ~ bands, respectively. The/9 band is a vibronic side band of the a band: i.e., while the c~ band is due to a pure electronic transition (with no change in vibrational quantum number, Qo-o), the fl band is due to the envelope of all the active vibronic transitions (in which the vibrational quantum numbers of the vibronically active normal modes increase from 0 to 1, Q, ~). These vibronically active normal modes "'mix" the ~ and Sorer transitions, thereby "'stealing" intensity from the Sorer band."' This is an example of the vibrationally induced intensity mentioned earlier. The characteristics of the rR spectra of heine proteins depend markedly on the exciting wavelength. The R scattering in resonance with the Soret and c~/3(or much weaker charge transfer transitions) is not the same and will be discussed separately. The scattering observed when excitation is in the cq3 region (see Fig. 17) will be discussed first. This is "'vibronic" scattering and can be described by the B term of Albrecht's theory. ~''~'' According to this theory, ":; H. C. Longuet-Higgins, C. W. Reclor, and J. R. Platt, J. Chem. Phys. 18, 1174 119501. "~ M. H, Perrin, M, Gouterman, and C. L, Perrin, ,I. Chem. Phys. 5(), 4137 (1969L

130

CONFORMATION; OPTICAL SPECTROSCOPY

[5]

those normal modes that are vibronically active in mixing the a and Soret transitions (those responsible for the /3 band) are expected to undergo resonance enhancement. The I_Land I~ components (see Fig. 2) of the R scattering obtained by Spiro and Strekas 9~ from the heme proteins ferrocytochrome c and oxyhemoglobin using 514.5 nm excitation are shown in Fig. 18. The spectra are dominated by bands in the 1100-1650 cm-' region, in good agreement with the predictions of Albrecht's theory, since the/3 band is centered about 1300 cm -~, above the a band. Furthermore, the bands in this frequency range are those due to the in-plane stretching of C--C and C--N bonds and the bending of C--H bonds,:'" which are capable of vibrationally mixing the in-plane a and Sorer transitions. The strong bands in the spectra of both ferrocytochrome c and oxyhemoglobin are all either inverse polarized (ip) or depolarized (dp). Only very weak polarized (p) bands at 1505, 1540, 805, and 695 cm -1 are evident in the spectrum of ferrocytochrome c and at 830, 790, and 676 cm-' in the spectrum of oxyhemoglobin. The presence of inversely polarized bands, possible only in resonance, is one of the novel features of the rR spectra of heine proteins. This phenomenon requires a class of vibrations with an antisymmetric scattering tensor. For vibronic scattering, the symmetries of the active vibrations must be contained in the direct product of the group-theory representations, F, of the a and Soret transitions (viz., those being mixed vibronically). Since these are both of E, symmetry F = FL,~,× FE. = F4,~ + FA~ + F~,o + Fs~

(30)

The expected polarizations of modes with each of these symmetry classes for the D4h point group can be deduced from their scattering tensors (tabulated by McClainH). The A~g and B2g modes are not vibronically active and, hence, are not resonance enhanced. The B~g modes are expected from group theory to be depolarized and the A2g modes, which have an antisymmetric scattering tensor (i.e., c~,u = -c~u~.) to be inverse polarized. Hence, the polarizations of the bands in this spectrum are consistent with the symmetry species of the vibrational modes which are expected to couple the a and Soret transitions. It should be pointed out that the values of the depolarization ratios of some of the bands in cytochrome c and other heine proteins exhibit dispersion:'7--i.e., they vary with excitation frequency. There also appear ~:' T. G. Spiro and T. C. Strekas, Proc. Natl. Acad. Sci. U . S . A . 69, 2622 (1972). "q"P. Stein, J. M. Burke, and T. G. Spiro, J. A m . Chem. S o t . . 97, 2304 (1975). 'rD. W. Collins, D. B. Fitchen, and A. Lewis, J. Chem. Phys. 59, 5714 (1973).

[5]

RAMAN A N D RESONANCE RAMAN SPEC1-ROSCOPY

131

[l

(A) FERROCYTOCHROME t

,

C

SO2-= 9 8 3 cm -~ -,,,.

~

~

~

o

o~

'-'2_

~

t

' T

Iu

I±

>-

g

I--

z ILl I"Z H

1700

i600

i500

1400

1300

1200

1/00

1000

900

800

700

600

(B) OXYHEMOGLOBCN

oT_;, Sl . '

£ .

I

~II~d

'P'PT-I

TI!?-P

-~'~ ~ ~°ll ~ ,-- ,.', 7 Vii ]1

I II ~ .

. . ~- ,'~ m ~ " ~ o

/IV T

~-

(~1

1500

1400

1300

II

dp

~o o ! c~ _ I dpdp tddDT ~ ~

-

-

16 0

o

dpdo ip~

^

O

~

-.---

1200

dpdp dp tO~ 0

,.o p p ~ 0 0 I

.

11

.,..,..~.,....,~._~ ~ , . . , l l00

1000

900

800

700

600

Av (cm-')

FIG. 18. Resonance Raman spectra of (A) ferrocytochrome c, 0.5 raM, obtained with excitation at 514.5 am, and of (B) oxyhemoglobin, 0.5 raM, obtained with excitation at 568.2 am. The scattering geometry is shown schematically in the diagram at the top. Both the direction and polarization vector of the incident laser radiation are perpendicular to the scattering direction. The scattered radiation is analyzed into components perpendicular (/_L) and parallel (L) to the incident polarization vector. Slit width, about 10 c m '. Adapted from T. G. Spiro and T. C. Strekas, Proc. Natl. Acad. Sci. U.S.A. 69, 2622 (1972).

132

CONFORMATION;

OPTICAL SPECTROSCOPY

[5]

.5 o

o

agl

470

500

550

580

,X. (nm)

FIG. 19. Excitation profiles for the prominent resonance Raman bands of ferrocytochrome c, and the electronic absorption spectrum ( ), both on a logarithmic scale. The points represent intensities of the indicated Raman band, measured relative to the vl sulfate peak from (NH4)2SO4 internal standard, with the available Ar+ and Kr+ laser lines. The profiles are displaced for clarity on an arbitrary log intensity scale. The available points were fit to the standard Gaussian curves displayed here by a DuPont 303 curve resolver. Adapted from T. G. Spiro and T. C. Strekas, Proc. Natl. Acad. Sci. U.S.A. 69, 2622 (1972). a n o m a l o u s l y p o l a r i z e d (ap) b a n d s - - t h o s e with PL v a l u e s b e t w e e n ¾ a n d ~. O n e p o s s i b l e e x p l a n a t i o n 4'97 for t h e s e p h e n o m e n a is that there exist accidental d e g e n e r a c i e s (two d i s t i n c t m o d e s u n d e r the same b a n d ) . A n o t h e r possibility is that the effective s y m m e t r y of the h e i n e g r o u p is lower than D4h. This c o u l d result in s c a t t e r i n g t e n s o r s with both s y m m e t r i c a n d ant i s y m m e t r i c c h a r a c t e r a n d e x p l a i n the a b o v e results. This e m p h a s i z e s the

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

133

need for caution when using the values of OL for rR bands obtained at a single excitation frequency for making symmetry species assignments. Excitation profiles covering the region of the /3 band of ferrocytochrome c ')'~ and the a band of oxyhemoglobin~) have been obtained and are shown in Figs. 19 and 20, respectively. The excitation profiles for all the bands of oxyhemoglobin peak at the frequency of the ~ (Qo-()) transition, u,, while those of the/3 band of ferrocytochrome c shift systematically to lower frequency with decreasing vibrational frequency.

I

!!

Au (cm-) ) o

755

• 1225

¢:

• )\t! .go Spec)~

V

P,

J¢l <~

D

_

1

l

I

1

500

550

600

650

X (nrn)

FIG. 20. Exitation profiles for the prominent resonance Raman bands of oxyhemoglobin and the electronic absorption spectrum (----), Measured relative intensities for the indicated Raman bands are plotted on a log scale and displaced for clarity. The dashed line marks the coincidence of the excitation profile peaks with the c~ peak of oxyhemoglobin at 577.6 nm, Adapted from T. C. Strekas and T. G. Spiro, J. Rarnan Spectrosc. 1, 387 (1973).

134

CONFORMATION"

OPTICAL SPECTROSCOPY

[5]

Their positions are in satisfactory agreement with the frequency of the various components of the/3 (Q0-0 transition, t, = t,~ + At,, where At, is the observed R shift. This is further proof that the observed rR bands are due to the same modes as those responsible for the vibronic components. Furthermore, this confirms the prediction made earlier that the intensity of a R band corresponding to a vibration that mixes two electronic states should peak at both the 0 - 0 (referred to earlier as the t,,.) and 0 - 1 (referred to earlier as the t,e + t,~j) frequencies. The observation of maxima at both the 0 - 0 and 0 - 1 frequencies of the same heme protein was not possible for either of the compounds of Figs. 19 and 20 owing to the limited availability of excitation wavelengths. However, double maxima in the cq~ region of the excitation profile of Mn(IlI)-etioporphyrin I have recently been observed. :~ The changes in the intensities of four of the prominent bands in the rR spectrum of ferrihemoglobin fluoride, as the excitation wavelength is scanned through the aft region and close to the Soret region, '~;'are shown in Fig. 2 I. The bands at 760 and 1175 cm -j, which are depolarized, and the band at 1340 c m - ' , which is inverse polarized, behave as expected for vibronically active modes in resonance with the fl band. The polarized band at 1373 cm -~, however, continued to increase in intensity as the Sorer band was approached. The frequency dependence of this band was measured accurately and shown '~'~to follow that expected for A-term [see Eq. (24)] scattering derived by Albrecht and Hutley. 2~ Furthermore, the depolarization ratio of this mode was within experimental error of the value ( ~ ) expected for a totally symmetric mode in resonance with the planar heine skeleton. '~'~ Hence, this band (and other weaker polarized bands at 1473 and 1587 cm -~) are due to A-term scattering in resonance with only the Soret transition. The utility of rRS for elucidating the features of electronic spectra can be shown from the data for ferrihemoglobin fluoride in Fig. 21. From the observed frequencies of the maxima of those bands in the excitation profile in resonance with t h e / 3 band (Q0-1), the frequency of the a band (Q0-0), which is not resolved in the visible absorption spectrum, can be calculated for each band. These frequencies are indicated by arrows in Fig. 2 I. These calculated frequencies are in excellent agreement with the frequency proposed for the o~band on the basis of low-temperature crystal spectra and confirm this assignment. The excitation profiles of other hemes '~'~'~°" have been used similarly to elucidate the origin o f bands in complex absorption spectra. "~J. A. Shelnutt, D. C. O'Shea, N. T. Yu, L. D. Cheung, and R. H. Felton,J. Chem. Phys. 64, 1156 (1976). '~'T. C. Strekas, A. J. Packer, and T. G. Spiro, J. Raman Spectrosc. l, 197 (1973). ""'S. Asher and K. Sauer, J. Chem. Phys. 64, 4115 (1976).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

135

XO \e

X,

A,, (cm-f ) ~e

1373

C:

.k\.

Cz

• it,

'

0-0 1340

t

jo-, \,3-o

0 IP

tie

o~,

/

7/o-,-0-0

7,0

0

Visible Spectrum

0 In t'%

--~ ~--Oo-o

t 450

I 500

I 550

I 600

X (nm) FIG. 2 I. Excitation profiles for the prominent resonance Raman bands of ferrihemoglobin fluoride and the electronic absorption spectrum. Exciting wavelengths are indicated by arrows at the bottom. Measured intensities are plotted on a log scale and displaced for clarity, for the indicated Ranlan bands. Arrows labeled Q, ~ mark the estimated intensity maxima, and those labeled Q,H, indicate the calculated position of the respective vibronic origin. Adapted from T. C. Strekas, A. J. Packer, and T. G. Spiro, J. Raman Spe('trosc. 1, 197 (1973).

The rR spectra of some heme proteins exhibit bands that are not due to the porphyrin ring. For eample, the rR spectra of oxy- and d e o x y h e m o globin obtained using excitation near the Sorer region show low frequency modes (not shown in Fig. 18) associated with the F e - N stretching motions, '"' in addition to porphyrin ring modes. These spectra are listed in Table X. The spectra of the oxy and deoxy forms have several obvious differences, especially in the region below 600 cm ' and in the frequencies of the bands near 1376 and 1223 c m - ' . The band at 567 cm -~ has been shown '°2 to be due to an Fe-O2 stretching mode. This band shifts to 540 "" H. Brunner and H. Sussner, Biochim. Biophys. Acre 310, 20 (1973). ""-'H. Brunner. Naturwissenschq(ten 61, 129 (1974).

136

CONFORMATION:

OPTICAL SPECTROSCOPY

[5]

TABLE X RAMAN FREQUENCIES AND ]NTENSITIES OF OXY- AND DEOXYHEMOGLOBIN SOLUTIONS"

Frequency t' (cm -*) OxyHb

DeoxyHb

Tentative assignment"

339 (0) 351 (1) 364 (2~ 380 (0)

v(Fe--N)

412 (1) 424 (I) 572 (1) 676 (3) 705 C0) 755 (3) 915 (0) 979 (I) 1001 C1) 1131 (2) 1173 (2) 1223 (2) 1282 (0) 1306 (0) 1376 (20) 1426 (0) 1468 (1) 1504 (2) 1551 (s) 1564 (s) 1587 (7) 1603 (0) 1636 (6)

673 (6) 708 (0) 755 (3) 915 (0) 979 (I) 997 (1) 1131 (2)

v(Fe--O~) or(ring) 0r(ring)

1173 (2)

1210 1228 1284 1306 1355 1394 1426 1472

(2) (s) (0) (0) (30) (s) (0) (2)

1549 1564 1590 1605

(7) (0) (7)

6(C-H)

v(==C-N)

(7) v(C===C) and v ( C ~ N )

" Adapted flom H. Brtmner and H. Sussner, Biochim. Biophys. Acre 310, 20 (1973). " Numerical figures in parentheses are relative peak intensities with that at 1355 cm ' taken as 30. The letter s denotes a shoulder. ' v denotes a stretching, c5an in-plane bending, and or an out-of-plane bending vibration.

cm -~ when 'nO._, is used, J"" and is not present in the spectrum of d e o x y h e m o g l o b i n . The enhancement of this axial-ligand m o d e probably involves scattering in resonance with a weak Fe---~ 02 charge transfer transition band, since an electronic transition polarized perpendicular to the plane o f the heine would be required to enhance such a mode.

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

137

The sensitivity of certain bands in the rR spectra of heroes to such perturbations as changes in oxidation and spin state is one of their important practical features. From the study of a large number of such systems, porphyrin ring modes that are sensitive to either the spin or oxidation state of the heme have been catalogued and are summarized elsewhere. ~~': These correlations can be used to deduce information about the spin states of less well characterized derivatives or to study the effects of interactions of heme groups with various molecules under biological conditions. The rR spectra of heine proteins exemplify the need to exercise caution when interpreting rR spectra. It is clear from the above discussion that rR spectra and their characteristic properties (such as the relative intensities and depolarization ratios of the bands) can depend markedly on the exciting wavelength. To minimize the possibility of misinterpretation due to such effects, a variety of excitation wavelengths should be employed and the effect of this variation on the spectra carefully studied. It should be clear that the interpretation of rR spectra is greatly facilitated by an understanding of the nature of the electronic transitions with which the scattering is in resonance. Nonheme Metalloproteins The interpretation of the rR spectra of several chromophoric nonheme metalloproteins will now be considered. Many metalloenzymes exhibit visible absorption bands which can be due to either metal-ligand charge transfer transitions (e.g., adrenodoxin) or to d--d transitions (e.g., co-carboxypeptidase A) localized on the metal. Only excitation under charge transfer transitions has so far produced resonance-enhanced R scattering. H e m o c y a n i n is a copper-containing oxygen-carrying protein found in the plasma of certain mollusks and crustaceans. The deoxy form is colorless, but the absorption spectrum of the blue oxy form, which contains the c o p p e r - o x y g e n complex, consists of an intense (ecu - 10~M -~ cm ~) band near 340 nm and a weaker (~cu - 500 M -~ cm -~) band near 570 nm,'":" Various assignments are possible for these bands. The identity of the ligands bound to the metal is not known. The R spectra obtained by Freedman et al. ~o:~for o x y h e m o c y a n i n from B,sycon canaliculaturn and Cancer rnagister using excitation at 530.9 nm are shown in Fig. 22. All but two of the peaks in each of these spectra (those at 749 and 267 cm ~ in B. canaliculatum and at 744 and 282 cm ' in C. magister) are also present in the spectra of the deoxy and copper-free forms (not shown). Furthermore, their intensities are independent of excitation frequency from 457.9 to 647.1 nm. Hence, these bands are not ~":~T. B. I;i-eedman, J. S. Loehr, and T. M. Loehr, J. A m . Chem. Soc. 98, 2809 (1976),

138

CONFORMATION" i

i

i

O P T I C A L SPECTROSCOPY

1

i

i

i

i

r

i

[5] i

749

[-Z

.-

I

I0

16 o

I

I

14oo

I

I

I

leoo

I

~ooo

1

I

8oo

I

t

6oo

A v ( c m -~)

FIG. 22. R a m a n spectra of o x y h e m o c y a n i n (A)Busycon c.naliculatum, 165 mg/ml, p H 9.8 and IB) Cancer magister, 60 mg/ml, pH 8.5. Slit width. 8 cm ': rate of scan, 20 cm-I/min; time constant. 5 sec. Peak at 932 cm ' is uffCIO4 ), and those at 1160 and 1520 are due to an impurity. Adapted from T. B. F r e e d m a n . J. S. Loehr, and T. M. Loehr, J. Am. Chem. Soc. 98, 2809 (1976).

resonance enhanced, and are due to the strong nonresonance R scattering of the aromatic side-chain and backbone modes of the protein. The bands at 749 and 267 cm -~ in B. canaliculatum and at 744 and 282 cm ~ in C. magister o x y h e m o c y a n i n s , on the other hand, are true rR bands. T h e y are absent in the spectra of the colorless deoxy- and apoproteins. Furthermore, the intensities of these bands do vary with excitation frequency, as shown by the excitation profiles in Fig. 23. Finally, the bands in the 700 c m - ' region, but not those in the 200 cm ' region, have been found to shift when ~sO., is substituted for "~O2, as shown in Fig. 24. On the basis of the a b o v e observations, the bands in the 700 cm -~ region have been assigned "':~ to the stretching vibration of the bound oxygen. The magnitudes of the reductions of these frequencies on going from ";Oe to ~O., strongly support this assignment. Since the values o f these oxygen stretching fi'equencies are in the same range as those found in peroxide c o m p o u n d s , it was proposed '°:~ that oxygen is bound as a peroxide ion O._,'-'- in o x y h e m o c y a n i n . The excitation profiles in Fig. 23B and 23E show that the resonance enhancement of these peroxide stretching bands essentially follows the absorption contour in the 570-rim region. This c o r r e s p o n d s to A-term (from Albrecht's theory) resonance enhancement in which there is vibrational interaction with a single excited electronic state. Since, for A-term resonance scattering, the vibrations that

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

139

undergo resonance enhancement are expected to be those that most nearly reproduce the distortions found in the excited state, it was proposed ""~ that the absorption band(s) in this region involves 02-'- --~ Cu(II) charge transfer. Such a transition would affect the strength of the O - - O bond and be expected to enhance the oxygen stretching mode. The frequency of the resonance-enhanced bands in the 200 cm-~ region are insensitive to 'sO2 substitution and, hence, do not involve motions of the oxygen atoms. The excitation profiles in Fig. 23A and 23D show that these bands are not in resonance with the 570 nm band, but rather with an absorption band at shorter wavelengths--probably the 345 nm band. C u imidazole complexes are known to have strong ultraviolet bands in the 310-360 nm range and C u - N stretching vibrations in the 250-290 cm

1.0

i

o

(A)

0.6 I

-\ -

(D)

o

0

o

0.2 ~

o

(E)

(8)

c

L4 I.O o

0.6 0.2

/

.__9~

a/

S

o

a/

_

I

l

I

i

I

° i

l

I

c)

I

(F)

c o

2 o

500

600

I

I

500

I

I

I

600

~. (nrn) FIG. 23. Excitation profiles for (A) the 282 cm ' band and (B) the 744 (O) and 704 cm (A) bands of Cancer magister h e m o c y a n i n whose absorption s p e c t r u m is shown in (C) and excitation profiles for (D) the 267 c m " band and (E) the 749 cm ' band of Busycon canaliculatum h e m o c y a n i n whose absorption s p e c t r u m is shown in (F). The symbols (~) refer to O h e m o c y a n i n and (©) to ~O: h e m o c y a n i n . The error s y m b o l s are avevage deviations for three or more experiments, Adapted from T. B. F r e e d m a n , J. S. Loehr, and T. M. Loehr, J. Am. Chem. Soe. 98, 2809 (1976).

140

CONFORMATION"

OPTICAL SPECTROSCOPY

Bus2con cana/iculatum ,

Hemocyanin

[5]

? J~

bz W bZ

H

Cancer rnaq/ster

Hemocyonin

,,A

I

/ (~

,oo U

I I I I I 800 700 600

~

I I I I 350 250

A ~ (crn -I ) FIG. 24. R e s o n a n c e R a m a n spectra o f "~O._,-and ~ O ~ - h e m o c y a n i n using 530.9 o r 457.9 nm excitation. C o n d i t i o n s are the same as Fig. 22. Adapted f r o m T. B. Freedman, J. S. Loehr. and T. M. Loehr. J. Am. Chem. Soc. 98, 2809 (1976).

region. Hence, these observations are consistent with both the assignment of the 267 and 282 cm -J rR bands to Cu-N (imidazole) vibrations of coordinated histidines and of the - 3 4 0 nm band in the absorption spectrum to a transition involving charge transfer between N (imidazole) and Cu(ll). While showing only two resonance-enhanced bands, hemocyanin illustrates the procedures necessary for detecting and interpreting resonance-enhanced R scattering from a complex metalloprotein. The "'blue'" copper proteins, which have been studied in different laboratories, "'4-''" and whose rR spectra show many and varied bands, "'~ O. Siiman, N. M. Young. and P. R. Carey, J. A m . Chem. Soc. 96, 5583 (1974). '"~' V. M i s k o w s k i , S. P. W. Tang, T. G. Spiro, E. Shapiro, and T. H. Moss, BiochemisoT 14, 1244 (1975). '"" O. S i i m a n , N. M. Young. and P. R. Carey, J. A m . Chem. S o c . 98, 744 (1976).

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

141

will now be considered, These proteins derive their name from their intense t E - 1300-5000 M -~ cm -~) absorption near 600 nm (which presumably involves some ligand + copper charge transfer), and which gives rise to their blue color, The structure of the Cu(ll) site responsible for this strong absorption and for the electron-transfer properties of these proteins is not known. The rR spectra of the five blue copper proteins ceruloplasmin, stellacyanin, laccase, plastocyanin, and ascorbate oxidase have been obtained ''''~ using excitation with three laser lines that lie in the vicinity of their 600-nm absorption band. These lines are shown in Fig, 25 together with the visible absorption spectrum of stellacyanin. The 200-600 c m ' region of the R spectra obtained by Siiman et al. '<"; using 647.1 nm excitation is shown in Fig. 26, and a listing of all the bands in the spectra of these five proteins, together with the tentative assignments, appear in Table XI. The spectral data and group vibrational assignments of these authors '''~ are in close agreement with those of other workers.'":' All the bands observed are resonance enhanced, polarized and have relative intensities that are independent of excitation wavelength, indicating that they are all in resonance with a single electronic transition (Albrecht's A term). Copper-ligand vibrational modes are expected from the g r o u p - m o d e concept to appear in the low frequency (<600 cm --~) region. Other higher frequency bands in the spectra (not shown in Fig. 26, but listed in Table XI) are probably due to ligand vibrational modes that are resonance enhanced. The identity of the ligands can be deduced from the observed frequencies by reference to the spectra of model copper-peptide complexes. The bands in the 350-429 c m - ' region have frequencies appropri-

o

o ..D

I 400

I

I 600

I 800

k(nm) FIG. 25. Electronic absorption spectrum of stellacyanin I--3.3 mg/ml, 0.05 M phosphate buffer, pH 5,5) showing the positions of the laser excitation lines. Adapted from O. Siiman, N. M. Young, and P. R. Carey, J. A m . Chem. Soc. 98, 744 (1976).

142

CONFORMATION: OPTICAL SPECTROSCOPY

[5]

g

(C)

t,= • I o

z uJ H

(D)

(E)

I

600

1

500

I

400

I

300

I -

zoo

ZX~(cm - I )

FIG. 26. Resonance Raman spectra of (A) ceruloplasmin, 11.6 mg/ml, in 0.05 M acetate buffer pH 5.5: (B) stellacynanin, 8.4 mg/ml, in 0.05 M phosphate buffer, pH 5.5; (C) laccase, 42.5 mg/ml, in 0.05 M phosphate buffer, pH 5,5; (D) plastocyanin, 1.5 mg/ml, in 0.05 M

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

143

ate for C u - - N (peptide bond or amide side chain) or possibly Cu--O (nonaromatic) stretching vibrations. The enhancement of ligand amide group vibrations near 1240 and 1660 cm -~ supports the existence of C u - - N (peptide) bonds. The band near 260 cm -1 can be assigned to either a Cu--S (cysteine) or C u - - N (imidazole) stretch. The former assignment is favored, since chemical evidence has implicated cysteine as a ligand and the band near 750 cm -~ could be an enhanced cysteine C--S stretching vibration. On the basis of the rR and other experimental data, models for the "blue'" copper site have been proposed, ~'~~1~'; Resonance Raman Labels

As an example of the use o f a rR label to study the mode of action of an enzyme, the interaction of the chromophoric ester substrate methyl 4-dimethylamino-3-nitro (c~-benzamido) cinnamate (symbolized aBAOMe) with papain is considered. This system has been studied by Carey and co-workers. "'7 Papain-catalyzed hydrolysis of esters proceeds through the formation of a covalent acyl-enzyme intermediate between the thiol group of the enzyme and the acyl group of the substrate. Carey et al."'7 were able to prepare and characterize a stable 4-dimethylamino-3-nitro (c~-benzamido) cinnamoyl-papain intermediate (c~BA-papain). The structures, absorption spectra, and rR spectra of the free substrate (c~BA-OMe), the acyl-enzyme (c~BA-papain) and the product of the enzymic hydrolysis [4-dimethylamino-3-nitro (c~-benzamido) cinnamic acid, symbolized by ~-BA-OH] obtained with 441.6 nm excitation are shown in Figs. 27-29, respectively. '"7 P. R. Carey, R. G. Carriere, K. R. L y n n , and H. Schneider, Biochemistry 15, 2387 (1976),

p h o s p h a t e buffer, pH 6.9: (E) ascorbate oxidase, 42.4 mg/ml, in 0.05 M phosphate buffer, pH 7.0. Experimental conditions: time constant, 5 sec: rate of scan, 30 cm '/rain" laser line, 647.1 nm.

A B C D E

Power (roW)

Slit width Icm ')

Sensitivity (cps)

80 80 60 40 30

10.0 7.0 7.0 7.0 7.0

2000 1000 2000 2000 1000

Adapted from O. Siiman, N. M. Young, and P. R. Carey, J. A m . Chem. Soc. 98, 744 ( 19767.

Z 0

0

0

0

L

0

i~ Z

R~

E

Z

~L~

Z

0

Z 0

CS~

~r~ ,~*

~t

I~

~ED

~..

[5]

RAMAN AND RESONANCE RAMAN SPECTROSCOPY

145

0

C --C --OMe I NH

I

Me2N

C:O }

NOz

Ph

(a- BA-OMe)

I

~

H C~

?,(9 C-- C ' " O

I

NH

Me2N

I

NO2

C-~-O

13" (a- BA-OH) O

H

C~

II

C-- C --S--Enz

I

N

Me2N/~

OX

NOz

TTT

( a -BA~papain)

FtG. 27. Structures of (I) methyl 4-dimethylamino-3-nitro(c~-benzamido) cinnamate and (11) 4-dimethylamino-3-nitro(c~-benzamido) cinnamic acid; and (111) the proposed structure for the acyl-papain enzyme. Adapted from P. R, Carey, R. G. Carriere, K. R. Lynn. and H. Schneider, Biochemistry 15, 2387 (1976).

T h e r R s p e c t r a o f t h e s u b s t r a t e (I) a n d p r o d u c t (11) a r e quite s i m i l a r . T h e s t r o n g d o u b l e t s n e a r 1620 c m - ' a r e d u e to - - C - - C - - a n d b e n z e n o i d ring m o d e s , a n d t h e b a n d s n e a r 1353 c m ' to the s y m m e t r i c s t r e t c h o f t h e nitro g r o u p . B a n d s d u e to v i b r a t i o n s o f t h e a - b e n z a m i d o g r o u p are not e n h a n c e d in t h e s e s p e c t r a . T h e s p e c t r u m o f t h e acy[ e n z y m e , on the o t h e r

146

CONFORMATION: OPTICAL SPECTROSCOPY

0.4

/

-

t

(~-BA-OMe)

/•I /~E

(a'-BA-OH)

f/'",.~/ /

".-."/

~p U C 0 r~ t,,. 0 o~

/

i/ I/

.

~111

/

',, ~

I

',, , ,

";

/

[5]

/

/

/

\

(a, BA-popain)

\\ \ \

0.2 N,,J

'~

\ ,,

I

300

I

\\

I

400

500

~k (nm) FIG. 28. Absorption spectra of the substrate (I), product (IlL and acyl-enzyme ( l i d whose structures are s h o w n i n Fig. 27.( ) ( 1 ) , - 1 . 5 x 10 ~ M i n 20% CH:~CN,80% H20, p H 6.0; ( . . . . ) 0 I L - 1.1 x l0 -s M in H20, p H 7.0; a n d ( . . . . ) (II1), - 1.4 × l0 -5 M in b o u n d a B A - O M e and 6.5 × l0 -:> M papain, pH 3.0, pathlength, l cm. Adapted from E R. Carey, R. G. Carriere, K. R. L y n n , and H. Schneider, Biochemistry 15, 2387 (1976).

hand, is quite different from that of either the substrate or the product. The absence of both the doublet near 1620 cm -~ and the band at 1353 cm -1, and the presence of a new, strong band at 1570 cm -~ should be noted. These profound spectral changes indicate that rather major changes in the structure of the chromophore have occurred. An examination of the R and rR spectra of azlactones, derived from aBA-OMe (Fig. 30), shows that the presence of the PhC--C--N--~CPh grouping correlates with the presence of an intense band near 1560 c m - ' , attributable to a high degree of ~,(C~N) character. On this basis, it was suggested that the 1570 cm -T mode of the acyl-enzyme is due to a stretching mode from a --C==C--N==C(--OX)-- grouping (proposed structure III in Fig. 27) formed by a rearrangement of the a-benzamido side chain. This interesting study illustrates the applicability of the rR technique to the study of substrate rearrangement in the active site. This technique provides the opportunity to study selectively the vibrational motion of those atoms and bonds that are expected to be important in the catalytic mechanism. The future for such studies appears bright.

[5]

RAMAN A N D RESONANCE RAMAN SPECTROSCOPY

147

m (a- B A - popoin)

o

03

z LLJ

,~

~

I(~-BA-OMe)

P'4

o

-

I

1800

L

L

t400

I

m

L

~000

A z.,(cm-~ ) FIG. 29. Resonance Raman spectra of species 1.11, and Ill whose structures are shown in Fig. 27. Conditions are as follows: (I) - 1 0 ~ M in 80% D._,O, 20% CD:~CN: 441.6 nm excitation: slit width, 9 cm- ~: power, 50 mW. (11) - 3 × 10 4 M in HeO, pH 13; 441.6 nm excitation; slit width, 9 cm ~; power, 50 roW. (111) Enzyme - 6 . 5 × 10 :' M, bound substrate - 1 . 4 × 10 :' M; 441.6 nm excitation; slit width, 9 c m - ' ; power, 50 roW. The letter "'s" denotes solvent (dimethyl formamide) bands. Adapted from P. R. Carey. R. G. Carriere, K. R. Lynn. and H. Schneider. Biochemistry 15, 2387 (1976).

148

CONFORMATION:

(A)

OPTICAL

-

0

A H

J

--

IS]

SPECTROSCOPY

•

if

NOz

C~O

Ph

(B)

Z Z H

(C)

f

Jeoo

i

I

J4oo

1

i

fooo

A u ( c m -q ) FiG. 30. Resonance Raman or Raman spectra of (A) compound I of Fig. 27 and of the two structurally related azlactones (B)4-(4-dimethylamino-3-nitro)benzylidene-2-phenyloxazolin-5-one and (C) 4-(2,4-dinitro)benzylidene-2-pbenyloxazoJin-5-one. All spectra are of solid samples in a KBr matrix and were taken using a rotating sample holder. Spectra CA) and (B) are resonance Raman spectra obtained with 441.6 nm excitation and (C) a nonresonance Raman spectrum obtained with 647.1 nm excitation. Adapted from P. R. Carey, R. G. Carriere, K. R. Lynn, and H. Schneider, Biochemistry 15, 2387 {1976).

Conclusion The R spectra of proteins can provide information about their structure and conformation. The backbone vibrational modes of the proteins can be correlated with the presence of the various topographical states, a n d t h e v i b r a t i o n a l m o d e s o f c e r t a i n s i d e c h a i n s , s u c h as c y s t i n e a n d

[6]

MAGNETIC CIRCULAR DICHROISM

149

tyrosine, can be correlated with their local conformation and environment. Changes in the R spectra of proteins, brought about by external influences, can also be used phenomenologically to study such processes as folding and unfolding. When R excitation falls in the region of an electronic absorption band, resonance e n h a n c e m e n t of vibrations associated with the c h r o m o p h o r e occurs. This selective e n h a n c e m e n t enables biological c h r o m o p h o r e s to be studied at low concentrations. Structural information may be derived from the vibrational bands found in rR s p e c t r a - - s u c h as the identification of ligands in c h r o m o p h o r i c metalloproteins, or the spin state of the heine in heine proteins. In addition, the behavior of bands in rR spectra, as the exciting wavelength is varied, can be used to elucidate the nature of the electronic transition(s) with which the scattering is in resonance. As laser sources in the ultraviolet b e c o m e available, the rR effect, which is presently applied almost solely to visible c h r o m o p h o r e s , will soon be expanded greatly in scope, and new classes of c h r o m o p h o r e will b e c o m e accessible to study. Acknowledgment The authors are indebted to Drs. A. C. Albrecht, G. Korenowski, F. R. Maxfield, and R. K. Scheule for comments and helpful discussions, to 1. Levine for proofreading, and to the many authors who have granted us permission to reproduce their results here. This work was supported by the National Institutes of Health, U.S. Public Health Service (GM-14312, GM-15003, CA-05292) and the National Science Foundation (PCM75-08691).

[6]

Magnetic

Circular Dichroism

By BARTON HOLMQUIST and BERT L. VALLEE In recent years renewed interest in the F a r a d a y effect has added a new dimension to spectroscopic examinations of biologically important molecules, First recognized in 1845,1 the F a r a d a y effect is the induction of optical activity by the application of a magnetic field. To a large extent the heightened interest is due to recent developments in both theoretical understanding and the instrumental capability to accurately measure magnetic circular dichroism (MCD): this, in turn, now enables the extensive application and the exploration of the technique to biochemical systems, The F a r a d a y effect originates from the effect of a magnetic field on the '-Faraday Diary," Vol. IV. G, Bell and Sons, Ltd.. London, 1933.