Low-temperature luminescence studies of proteins and related molecules

2 LOW-TEMPERATURE LUMINESCENCE STUDIES OF PROTEINS A N D RELATED MOLECULES J. N . MILLER

Loughborough University of Technology, Loughborough, Leicestershire CONTENTS 43

I. INTRODUCTION

43

II. THEORY

46

III. EXPERIMENTAL

A. B. C. D.

46 49 50 51

Instrument Modifications Solvent Systems Luminescence Spectra Phosphorescence Lifetimes

IV. LUMINESCENCEOF AMINO ACIDS AND PEPTIDES

A. B. C. D.

Introduction Tyrosine and Tyrosine-containing Peptides Tryptophan and Tryptophan-containing Peptides Peptides containing Tyrosine and Tryptophan

V. PROTEIN LUMINESCENCE

A. B.

Class A Proteins Class B Proteins 1. Introduction 2. Luminescence Properties of Typical Class B Proteins 3. Tyrosine Luminescence in Class B Proteins 4. Class B Proteins showing Unusual Tryptophan Luminescence

52 52 53 55 57 58 58 6O 6O 6O 61 63 65

VI. CONCLUSIONS

65

VII. ACKNOWLEDGEMENTS

66

VIII. REFERENCES

41

2 LOW-TEMPERATURE LUMINESCENCE STUDIES O F PROTEINS A N D R E L A T E D MOLECULES J. N. MILLER

Loughborough Universityof Technology, Loughborough,Leicestershire I. INTRODUCTION In the twenty years since the first systematic studies were published, the intrinsic luminescence properties of proteins have been increasingly used as powerful tools for the investigation of the structures and interactions of these molecules. Much detailed information has been amassed on the conformation of proteins, their susceptibility to denaturation, and their interactions with other proteins, nucleic acids, and low molecular weight organic and inorganic species. Whereas fluorescence measurements at room temperature provide unique opportunities for the study of changes and interactions occurring during the lifetime of the singlet excited state of the molecule, at low temperatures (normally 77K) bimolecular processes are virtually absent. The dynamic effects of solvent and other molecules can thus be distinguished from those arising directly from the native structure of the protein. Furthermore, phosphorescence as well as fluorescence can be observed at low temperatures: a number of additional parameters such as phosphorescence lifetime and fluorescence: phosphorescence ratio, which may be structure-dependent, are thus available for study. A number of the early papers (e.g. Debye and Edwards, 1952) show that the advantages of recording luminescence spectra at low temperature were appreciated from the first, but for some time such investigations were performed using specially designed apparatus. Many contributions originated in the U.S.S.R. and have been extensively reviewed by Konev (1967) and by Vladimirov (1965). More recently, the introduction of low-temperature attachments for the commercially-available spectrofluorimeters has brought the technique within the capacity of many laboratories and there has been a corresponding increase in the number of applications described in the literature. In this survey the theory and practice of low-temperature luminescence measurements are outlined and recent applications to protein structural studies are critically reviewed. For discussions of wider aspects of protein luminescence, with the emphasis on results obtained at room temperature, the reader is referred to the detailed review by Longworth (1971) and the more concise annual reviews of Brockleburst (1970, 1971, 1972). II. THEORY Figure 1 shows the principal luminescence processes encountered in simple molecules. The absorption of a quantum of radiation, which takes about 10-15 s, normally promotes a molecule from the ground electronic state, So, to an excited singlet state ($1, $2, etc.) and possibly also to an excited vibrational state. Within 10-12 s the excess vibrational energy is dissipated as heat (internal conversion) until the molecule occupies the ground vibrationa 43

44

J.N. MmLER

state (v' = 0) of the lowest excited singlet state $1. Even if the original absorption promotes the molecule to $2 or a higher singlet state, the overlap of the vibrational levels of the excited singlets ensures that energy is dissipated until S~ is occupied. Thus the wavelength of the fluorescence emission process (S~ ~ So)I" will normally be greater than the wavelength of absorption, and will be the same whether the short- or long-wavelength absorption bands of the molecule are employed in its excitation. The internal conversion S~->So is also feasible, but much less likely than internal conversions involving higher singlet states. The lifetime of fluorescence, ~-r, is the time required for the number of molecules in a sample occupying Sx to fall to 1/e of its initial value: it is thus the reciprocal of the first order rate constant of the fluorescence process if non-radiative deactivation processes are absent.

?

l

S 4 :S 2 t V::0

t

::s,

~

,,

Energy

6 S 4 3 2 1 V:O

J So

Fie. 1. Luminescence processes in a simple molecule. Straight lines represent transitions in which radiation is absorbed or emitted; corrugated lines represent radiationless transitions. A, absorption; F, fluorescence; P, phosphorescence; IC, internal conversion; ISC, intersystem crossing. V and V' values define the vibrational energy levels in the ground and first excited electronic states respectively. Fluorescence lifetimes are normally of the order of 10 ns. They can be measured directly on commercially-available equipment (Meserve, 1971): such instruments are, however, distinct from conventional spectrofluorimeters, and the intrinsic fluorescence lifetimes of proteins have not yet been widely studied. The lowest triplet state Tt can be populated via the intersystem crossing process St,~÷T1. The rate constant for this process ( ~ 108 s- t) enables it to compete with fluorescence as a possible route for the depopulation of St. Since the rate constant for intersystem crossing is so much lower than that of internal conversion processes in the excited singlet states, it is only significant at the St ~ ÷ T t level. Phosphorescence is the radiative transition 7"1 -->So. Since 7"1 normally has a lower energy than St (Hund's rule), the wavelength of phosphorescence of a given molecule is longer than the wavelength of fluorescence. The "forbidden" nature of the phosphorescence transition results in very long phosphorescence lifetimes, ~-p, t Throughout, ~ indicates a radiative transition, ~ ÷ a non-radiative one.

Low-TEMPERATURE LUMINESCENCE STUDIES OF PROTEINS AND RELATED MOLECULES

45

of 10-3-102 s. At room temperature the/'1 state normally suffers collisional deactivation in this long period, and no phosphorescence is seen; thus phosphorescence measurements are conventionally performed at 77K. In these conditions phosphorescence lifetimes can be measured without difficulty (see below, section IIID). It has recently been reported (Schulman and Walling, 1972, 1973) that charged organic species, adsorbed on solid surfaces in completely dry conditions, exhibit phosphorescence at room temperature, but it seems doubtful whether this technique will be of value in protein studies. The non-radiative internal conversion process/'1,~,÷So also competes in the depopulation of T1. Phosphorescence phenomena are thus regulated by two sets of processes: those that control the intersystem crossing rate constant, and those that affect the loss of energy from T~ itself. Other luminescence phenomena such as delayed and sensitized fluorescence are reviewed by Parker (1968): they are not of great significance in protein studies. The relative significance of the various processes available for restoring an excited molecule to the ground state is expressed quantitatively as quantum yields. Thus the quantum yield of fluorescence, 4'v, is that fraction of excited molecules which return to So via the fluorescence mechanism. It can be shown by application of steady-state kinetics that ~/~ is a function of the rate constants of the processes S~ -+ So (fluorescence), $1 ~'÷So (internal conversion), Sa,~v÷T1 (intersystem crossing), and of bimolecular quenching processes. Similarly the quantum yield of phosphorescence, ~p is a function of the rate constants of/'1 So (phosphorescence), 7"1~,÷S~ (reverse intersystem crossing), T~~ - S o (internal conversion), of bimolecular processes, and of the quantum yield of triplet formation, q~r. It is noteworthy that phosphorescence lifetimes are independent of ~r: thus a change in the quantum yield of phosphorescence without a concomitant change in Zp is quite possible, and indicates that the intersystem crossing rate has been affected. The measurement of absolute quantum yields, recently reviewed by Demas and Crosby (1971), is far from simple. Many published data rely on assumed fir values of "standard" molecules such as quinine sulphate (see below, section IIIC): the dangers of this procedure are obvious (Fletcher, 1969). Phosphorescence quantum yields obtained at 77K are even more suspect because of the optical artifacts that occur at this temperature. The ratio ~r/~p can, however, be obtained with reasonable precision at 77K (see below, section IIIC) and may be of value in protein studies. The virtual absence of bimolecular processes at low temperature (see, however, Froehlich and Morrison, 1972) may produce a considerable increase in ffF values, compared with those obtained at room temperature. It is thus not permissible to obtain ~p values by combining room temperature ~F values with ffF/ffe ratios obtained at 77K. In complex molecules containing more than one potentially luminescent centre, intramolecular energy transfer may occur. If the emission band of one luminophor (the donor) overlaps the absorption band of another (the acceptor), then excitation of the donor followed by radiationless energy transfer may result in emission from the acceptor being observed. If the transfer process is highly efficient, emission from the donor may not be observed at all, but in other cases the energy transfer may be only partially effective, in which case excitation and emission spectra will contain contributions from both centres. Energy transfer processes may be effective over distances comparable with the diameters of many proteins (F6rster, 1959; Steinberg, 1971) and may have important effects on the luminescence behaviour of these molecules.

46

J.N. MILLER

III. EXPERIMENTAL

A. Instrument Modifications The optical layout of a conventional spectrofluorimeter (Fig. 2) is too well known to require detailed description. Light from an intense source (usually a high-pressure xenon discharge lamp) passes to the sample by way of a grating monochromator. Luminescence signals pass through a second monochromator and are detected at right angles to the incident beam by a standard photomultiplier tube. The photomultiplier output is displayed, after appropriate amplification, on a meter or chart recorder. A shutter between the light source and the sample permits the latter to be protected from radiation except when measurements are actually in progress. It is particularly important to use this shutter in protein studies, Excitation side Source

Monochromator/ filter

~

Sample

hosphoroscope f required) Emission side Monochromator / filter

Readout

P.M.

FIG. 2. Schematic layout of a fluorimeter/phosphorimeter.

since ultraviolet radiation has a number of deleterious effects on protein molecules. Tryptophan residues are modified to give a derivative, possibly N-formyl kynurenine (Savige, 1971), whose fluorescence overlaps the phosphorescence band of unmodified tryptophan, and in extreme cases peptide bond cleavage may also occur (Wilson and Foster, 1970). Since low-temperature luminescence spectroscopy should be regarded as a technique which complements rather than replaces room temperature fluorescence measurements, it is important that any modifications to the spectrofluorimeter which are necessary for lowtemperature work should be as simple as possible and readily reversible. In practice these modifications are normally confined to the sample compartment, and can be completed in 10-15 min. They have two distinct functions: firstly, to permit the sample to be cooled using liquid nitrogen and, secondly, to allow the prompt emission of fluorescence (and scattered light) to be distinguished from the much longer-lived phosphorescence emission. Figure 3 shows two of the cooling devices that have been used. The commonest type (Fig. 3a) consists of a partially silvered silica Dewar flask, whose unsilvered stem (outside diameter ~ 12 mm) lies in the fluorimeter light path. The sample is contained in a spectrosil

LoW-TEMPERATURELUMINESCENCESTUDIESOFPROTEINSANDRELATEDMOLECULES

47

tube (typical dimensions: outside diameter 4 mm, inside diameter 2 ram, sample volume 0.1-0.2 ml) which is mounted in the liquid nitrogen-filled Dewar. This relatively straightforward arrangement suffers from a number of disadvantages. The light beam has to pass through several extra thicknesses of silica, and also through the coolant. If the inside of the Dewar and the outside of the sample tube are not kept scrupulously clean and free of scratches, the liquid nitrogen bubbles freely and contributes a flickering component to the observed signal. Furthermore, the sample tube, which may be over 20 cm long and is normally supported close to its upper end, may be difficult to position reproducibly: in quantitative work, coefficients of variation may exceed 10 ~o. By mounting a small motor above the spectrofluorimeter sample compartment, however, and rotating the sample tube at speeds of a few hundred rpm this figure can be considerably reduced (Hollifield and Winefordner, 1968; Gifford et al., 1972).

--

- L i q u i d N2

Sample tube-

.Silvering (a) ~_

Sample

/Quartz Dewar

t l

tube

hi II

!!l .

,I

__ _.iqo,...

{b)

,ri

~

Frost shield

FIG. 3. Coolingsystemsused in low-temperatureluminescencestudies. The second cooling system in common use is shown in Fig. 3b. In this case the sample is contained in a shorter silica tube which is cooled by conduction using a metal cold finger and a liquid nitrogen reservoir. Many of the disadvantages of the first system are thus removed, but they may be replaced by the difficulty of ensuring good thermal contact between the sample tube and the cold finger. The sample may thus be cooled at an inconveniently slow rate. It should be noted that although it is generally stated that measurements are made at 77K, the actual sample temperature is likely to be higher: a value of ~ 100K has been given (Weinryb and Steiner, 1970). At temperatures below that of the solvent glass transition, however, small variations in sample temperature are unlikely to have much effect on ~p values. Whichever cooling system is used, it is normally necessary to blow a stream of dry gas

48

J.N. MILLER

over the portion of the Dewar or sample tube that is in the light beam. This effectively prevents frost formation on its outer surface. Short-lived and long-lived light signals are distinguished by means of a phosphoroscope (light chopper). The most common type consists of a small rotating cylinder, which surrounds the Dewar flask stem or sample tube, and is driven by a small motor mounted beneath the sample compartment. The cylinder has two or more vertical slots cut in it so that when the sample is being excited by the light source, no light can reach the detector (Fig. 4). After the cylinder has turned through 90 °, however, some of the long-lived phosphorescence signal remains and is measured. In the latter position no light is reaching the sample, so no fluorescence is observed. Thus only phosphorescence (and other delayed emissions) can be observed when the phosphoroscope is in the beam: when it is removed total luminescence, i.e. fluorescence plus phosphorescence is detected. The exact sequence of events in the course of one complete cycle of the phosphoroscope is described in detail by O'Haver and Winefordner (1966), who have also discussed the Becquerel phosphoroscope. This device utilizes two rotating discs in place of the slotted cylinder. A single disc phosphoroscope (Hollifield and Winefordner, 1969) and a system using a pulsed light source (Fisher and Winefordner, 1972) have also been described, but neither seems readily adaptable to conventional spectrofluorimeters. Sample Source

/

~"~ Can

i

I---lOeector

Incident light

F-q

Prompt emission (fluorescence)

Delayed emission (phosphorescence)

FIG. 4. Operation of a rotating can phosphoroscope. The rotating cylinder phosphoroscope has the disadvantage that a substantial fraction of the phosphorescence is lost because it is interrupted by the cylinder. The exact fraction of light lost depends on the dimensions of the cylinder and the number and size of the slots in it, but it may exceed 90 % of the total phosphorescence emission. The ingenious phosphoroscope described by Longworth and Bovey (1966) comprises two tuning forks, one in the exciting beam and one in the emitted beam, which are used in conjunction with a phase-sensitive amplifier. When the excitation tuning fork is vibrating only the in-phase fluorescence is detected; if the two tuning forks are operated 180 ° out of phase only phosphorescence will be observed; and when only the emission tuning fork is used, total luminescence will be recorded. In addition to the advantages of phase-sensitive detection, this system places no constraint on the design and dimensions of the cooling arrangements.

Low-TEMPERATURE LUMINESCENCESTUDIES OF PROTEINS AND RELATED MOLECULES

49

B. Solvent Systems Until recently it was believed that solvents used for luminescence spectroscopy at 77K should produce a clear rigid glass on rapid cooling, and a considerable number of suitable systems has been listed (Winefordner et al., 1967). In studies of proteins and related molecules, however, there are further requirements: the solvent chosen should also be hydrophilic, so that conformational changes in proteins are minimized, the proteins should be reasonably soluble in it, and luminescent impurities should be at a minimal level. Aqueous solutions form opaque snows when cooled to 77K, and they tend to cause shattering of the sample tubes, because of the large expansion and contraction effects. Amino acids and proteins are thus commonly studied in solvents such as ethanediol: aqueous buffer 1:1 v/v or propanediol:aqueous buffer 1:1 v/v. These mixtures normally cool to give glasses which are clear or show only a few cracks. Nevertheless, the variation in the appearance of the glass and the resulting effects on the magnitude of light scattering and other phenomena must contribute substantially to the poor reproducibility of low-temperature measurements, and again the rotation of the sample cell may improve results. Cracks and imperfections in the glassy solid are minimized by the use of narrow-bore tubes. It has been suggested that the addition of moderate concentrations of sugars to aqueous solutions may also produce clear glasses (Shaklai and Daniel, 1972). The assumption that aqueous solutions containing polyhydroxy compounds produce no substantial changes in protein conformations is not without precedent, since solutions containing sucrose or glycerol are widely used in studies of the depolarization of fluorescence of dye-protein conjugates at room temperature. It has also been established (J. N. Miller, unpublished work) that the conformation-dependent near ultra-violet optical rotatory dispersion of bovine 9,-globulin is the same in a solvent containing 50 ~ glycerol as in buffered aqueous solution. Water which has been distilled several times from a glass or silica still is likely to be more or less free of luminescent impurities, but ethanediol and propanediol often contain undesirable contaminants. The concentration of these may vary widely from one batch of solvent to another, and may increase after a bottle has been opened, unless the liquid is stored under nitrogen. Solvents and other materials which are non-luminescent or feebly luminescent at room temperature may be strongly luminescent at 77K, and extreme care is required to minimize interferences from such sources. Sample capillaries also contribute to the background signal at 77K. Their luminescence may be partially polarized, in which case it can be minimized using a polarizing film (Lukasiewicz et al., 1972a). Sample tubes are best cleaned with the aid of concentrated nitric acid or an ultrasonic cleaning bath: the use of detergents is disastrous, as most are strongly fluorescent. In an alternative sampling system, amino-acids and proteins can be studied by embedding them in thin glass-supported films of polyvinyl alcohol, which is water-soluble, exhibits little background luminescence and may be used at temperatures as high as 200K. This method has recently declined in popularity, possibly because it is less suited to the optical systems of conventional fluorimeters. A most promising development is foreshadowed by the recent publications of Winefordner's group (Lukasiewicz et al., 1972a, b, c) which have demonstrated that lowtemperature measurements can be made successfully in aqueous solutions containing low concentrations of salts. These authors have employed open-ended, thick-walled capillaries (internal diameter lmm, external diameter 6 mm) to resist expansion and contraction effects, and have averaged out optical inhomogeneities in the snowed matrix by rotating the

50

J . N . MILLER

sample tube as previously described. The tubes are easily filled by surface tension (sample volume ~ 20/~1), and the aqueous solvent has the additional advantages of low luminescence background, and straightforward control of pH. The intensity of phosphorescence signals is critically dependent on the salt concentration, presumably because of the effects of the latter parameter on the physical properties of the matrix. The system has thus far been applied only to the quantitative determination of a few low-molecular-weight species, but it seems likely that it will also be of great value in protein studies. It is well established (Zander, 1968) that the addition of "heavy atoms" (e.g. Br, I, metal ions) to luminescent systems causes an increase in the intersystem crossing rate constant, and a consequent quenching of fluorescence: such effects may constitute a useful probe of protein conformation (Lehrer, 1967). At low temperatures, where the enhanced rate of intersystem crossing may also be manifested as an increase in ~e, the most satisfactory reagent to use for this purpose in protein work is probably potassium bromide (King, 1972; cf. McCarthy and Dunlap, 1970).

C. Luminescence Spectra Excitation and emission spectra obtained from many spectrofluorimeters are said to be "uncorrected", i.e. they reflect the properties of optical components of the instruments as well as those of the sample. Thus an excitation spectrum, obtained by scanning the excitation monochromator at a fixed emission wavelength, is a function of the wavelength dependence of the light source energy output, and of the light intensity transmitted by the excitation monochromator. (The gratings used in excitation monochromators are usually blazed for maximum transmission at ~ 300 nm.) An emission spectrum, on the other hand, is obtained by scanning the emission monochromator at a fixed excitation wavelength, and is thus affected by the wavelength dependence of the transmission properties of the emission monochromator (maximal at ~ 450 nm) and of the response of the photomultiplier tube. Such effects may result in the apparent wavelengths of maximum excitation and fluorescence of proteins being 10-15 nm from their true values: phosphorescence spectra occur in a region where the photomultiplier response is only slightly dependent on wavelength, and are thus less affected. Correction procedures (Chen, 1967; Udenfriend, 1969) are tedious, and many workers still publish uncorrected spectra, thus preventing authentic comparisons of results obtained on different spectrofluorimeters. However, a number of commercially-available instruments now have built-in correction facilities, and the increased use of such equipment will be of great benefit. Corrected spectra are of particular value in the determination of relative or absolute quantum yields. It may be shown that if dilute solutions of two substances, 1 and 2, are observed in identical conditions, then the ratio of their quantum yields is given by

(4~)~ (~F)I

A~ (ODh al (OD)2

where A~ and A2 are the areas under the corrected fluorescence spectra (plotted as fluorescence intensity against frequency) and (OD)I and (OD)2 are the optical densities of the two samples at their respective excitation wavelengths. Of course if one of the substances is a standard of known (or assumed) quantum yield, then the other quantum yield can easily be calculated. A similar expression applies to phosphorescence phenomena. If the two compounds being compared are chemically similar and have similar excitation

LoW-TEMPERATURELUMINESCENCESTUDIESOFPROTEINSANDRELATEDMOLECULES

51

and fluorescence wavelengths and fluorescence bandwidths, then a reasonable approximation to (ff~)2/(ffe)l can he obtained from uncorrected spectra, using fluorescence intensities rather than spectral areas. Fluorescence:phosphorescence ratios of similar molecules can also be estimated from uncorrected spectra. Thus the ratio c/,r/c~p for a tryptophancontaining molecule at 77K can be obtained by comparing its uncorrected total luminescence spectrum with that of tryptophan itself, obtained in the same conditions. Since, at 77K, the wavelengths of fluorescence and phosphorescence of most tryptophan-containing species are very similar, and since the ratio ffe/~e for tryptophan itself is known (see below), the fluorescence: phosphorescence ratios of tryptophan derivatives can quickly be determined. I

I

I

I

i

2

3

4

s

6

log

0

1

seconds

FIG. 5. Determination of phosphorescence lifetimes: the upper graph is a smoothed experimental decay curve, and the lower one a semi-logarithmic plot of the results.

D. Phosphorescence Lifetimes The possibility of measuring phosphorescence lifetimes constitutes one of the main advantages of low-temperature luminescence measurements, and a number of techniques have been devised for this purpose. The phosphorescence lifetimes of proteins and related molecules almost invariably exceed 1 s, and the simplest way of measuring them is to make a direct record of the phosphorescence decay that occurs when a shutter interrupts the exciting light, using a fast-response strip-chart recorder. The recorder trace is then converted to a semilogarithmic plot (Fig. 5) and the lifetime obtained from the slope of the resulting straight line. The observed phosphorescence decay of a complex molecule may represent the sum of a number of components each with its own characteristic lifetime. If these lifetimes are sufficiently distinct, however, the use of the semi-logarithmic plot allows their separate

52

J.N. MILLER

determination, and the relative intensities of the individual components of the phosphorescence can also be estimated (Kuntz, 1971). The latter method has found applications in analytical chemistry, under the title "time-resolved phosphorimetry" (St. John and Winefordner, 1967). A rotating-cylinder phosphoroscope, whose speed has been calibrated stroboscopically, can also be used for the direct determination of phosphorescence lifetimes, as any given speed of rotation corresponds to a fixed delay time (Fig. 4). Constant speeds below a few hundred rpm are difficult to maintain, however, and the method is therefore only suitable for the determination of millisecond lifetimes. Lifetimes between ~0.05 and 1 s are best determined by recording the phosphorescence decay on a calibrated storage oscilloscope, and expressing the results semi-logarithmically as before. IV. L U M I N E S C E N C E OF AMINO ACIDS AND PEPTIDES A. Introduction

The luminescence of proteins is dominated by the contributions of two aromatic amino acids, tyrosine and tryptophan, and much of the remainder of this review will deal with the properties of these molecules and their derivatives. In this section the luminescence of the amino acids themselves and of some simple peptides in which they are incorporated will be discussed, in view of their potential interest as model systems. A third amino acid, phenylalanine, is also luminescent (AAt ---- 258 nm; Art -----282 rim; Ap1- = 385 nm; ~'e = 5.5 s), but its molar extinction coefficient (e2ss,m ---- 190) and its fluorescence quantum yield at room temperature (~r "~0.03) are so small that its contribution to protein fluorescence, when it occurs along with tyrosine and tryptophan, is often considered negligible (Teale and Weber, 1957). At 77K, however, the total luminescence quantum yield of phenylalanine, ~F ~-~e is close to unity (ffF ---- 0.41, ~, = 0.59; Bishai et aL, 1967). This does not necessarily imply that phenylalanine luminescence should be observed in proteins at 77K since, when the amino acid is incorporated into a polypeptide chain, energy loss by SI,~÷So internal conversion may occur, as is apparently the case with poly-L-phenylalanine (Longworth, 1971). Furthermore, since the fluorescence emission band of phenylalanine overlaps the absorption bands of tyrosine and tryptophan, radiationless energy transfer to the latter amino acids is possible. Finally, the low extinction coefficient of phenylalanine will again result in only feeble luminescence. It is thus not surprising that, although luminescence has been detected from poly-L-phenylalanine, and in certain phenylalanine-containing dipeptides (Weinryb and Steiner, 1968) and cyclic peptides such as polymixin B and gramicidin S (Longworth, 1971), which contain no tryptophan or tyrosine, phenylalanine luminescence in proteins has been reported in only one case. Grimes et al. (1969) showed that collagen, which contains phenylalanine and tyrosine but not tryptophan, exhibited phenylalanine phosphorescence, both at 77K and when in the form of a glassy solid at ambient temperatures. Possibly the unusual triple-helix structure (cf. poly-L-proline II) of this molecule minimizes energy transfer and other energy dissipation processes. It has recently been reported (Tatischeff et al., 1973) that histidine is feebly fluorescent (Aa -----210 nm, Ar -----300 nm) in neutral solution at room temperature, but it is not apI"Throughout, ~,t refers to the wavelengthof maximum excitation, )it to the wavelength of maximalfluorescence, and h~ to the wavelength of maximal phosphorescence.

Low-TEMPERATURELUMINESCENCESTUDIESOF PROTEINSAND RELATEDMOLECULES

53

parent whether the effect can be observed when this amino acid is incorporated in a polypeptide chain, or whether histidine is phosphorescent.

B. Tyrosine and Tyrosine-containing PeptideJ Figure 6 shows the excitation and luminescence spectra of tyrosine at neutral pH. The two principal absorption bands are at 222 nm (~ = 9000) and 275 nm (E ---- 1400) (Beaven and Holliday, 1952). In practical measurements, the high-wavelength band is used almost exclusively. At room temperature, the fluorescence maximum occurs at ca. 303 rim. At 77K this maximum may shift slightly to lower wavelengths, although uncertainties arise because of difficulties in correcting spectra in this region. The phosphorescence maximum is at 387 nm and the phosphorescence lifetime 2.6-2.8 s. Debye and Edwards (1952) and I

11 200

I

Tyroslne

aoo

4oo

so6 ~,,nm

FIG. 6. Absorption (A), fluorescence(F) and phosphorescence (P) spectra of tyrosine in neutral solution. King (1972) report that the phosphorescence band is devoid of fine structure, but spectrum published by Longworth (1966) shows a number of subsidiary maxima: such effects may be solvent dependent. The quantum yield of tyrosine fluorescence at room temperature is 0.21, but at 77K it increases to 0.47 and the total luminescence quantum yield is unity (ffp ----0.53; ~e/ffp = 0.89). At p H 0 the fluorescence quantum yield is reduced, both at room temperature and at 77K, although the total luminescence quantum yield at the latter temperature remains 1 (fir = 0.34; ~be = 0.66; ~ / ~ p = 0.52) (Bishai et aL, 1967). Ionization of the hydroxyl group of tyrosine (pKa = 10) produces considerable changes in the absorption and luminescence spectra. The absorption maxima shift to 240 nm (~ = 11,000) and 293 nm (E = 2300), and the wavelengths of maximal fluorescence and phosphorescence also undergo bathochromic shifts. The fluorescence maximum is at 340-345 nm at room temperature, and 315-325 nm at 77K: the discrepancy may arise from solvent effects, such as the formation of exciplexes. The phosphorescence maximum is at ca. 400 nm

54

J.N. MILLER

and the phosphorescence lifetime falls to 1.2-1.4 s. The fluorescence:phosphorescence ratio at 77K falls dramatically (~r = 0.14; ~p = 0.86; ~ F / ~ = 0.16), and the fluorescence quantum yield at room temperature is so low (~r = 0.005; Bishai et aL, 1967) that some authors, e.g. White (1959), have stated that ionized tyrosine is non-fluorescent. In view of the use of iodide ions in studies of fluorescence quenching of proteins at room temperature, the effects of "heavy atoms" on amino acid luminescence are of interest. Bromide and iodide ions at concentrations up to 3 M decrease the ~r/~p ratio of tyrosine to ca. 0.5 : the phosphorescence lifetime falls to approximately 1.8 s, although the decay no longer obeys first-order kinetics. The phosphorescence decay of the tyrosinate ion is similarly altered, but there is no parallel change in the luminescence spectra. In contrast, 3-iodotyrosine and thyroxine are totally non-luminescent (King, 1972). Incorporation of tyrosine into a homopolypeptide has a number of effects on its luminescence properties. Poly-L-tyrosine, which has an a-helical structure with the planes of the aromatic rings perpendicular to the helix axis (Brady and Salovey, 1967), is hypochromic, and its quantum yields, both at room temperature (~r =0.07) and at 77K (~p -----0.35; ~, = 0.15; ~ / ~ e = 2.33) are lower than those of tyrosine. However, the wavelengths of maximum fluorescence and phosphorescence and the phosphorescent lifetime are similar to those of the free amino acid (Longworth, 1966). These data suggest that the energy loss at 77K (~r + ~e = 0.50) which occurs when tyrosine is incorporated into a polypeptide may be due to a reduction in the intersystem crossing rate and the occurrence of SI-,,+So internal conversion. Ionized poly-L-tyrosine, a disordered polypeptide, has rather similar fluorescence properties to those of ionized tyrosine, but its phosphorescence lifetime (~-p -0.9 s) and fluorescence:phosphorescence ratio (~r/~e = 0.10) are further reduced. As the pH of a solution of poly-L-tyrosine is raised, it retains its a-helical conformation until the degree of ionization reaches 0.45 (Fasman et al., 1964). Since the luminescence properties of tyrosine and its anion are quite different, such a system provides an ideal opportunity for studying the effects of tyrosinate ions on neighbouring tyrosine residues. It has been established (Longworth and Rahn, 1967) that efficient singlet energy transfer occurs from tyrosine to tyrosinate, both at room temperature and at 77K. The presence of as little as 3 % of tyrosinate in the polymer caused a 50 % reduction in the fluorescence quantum yield of tyrosine. Fluorescence polarisation measurements indicate that hot~ tyrosine-tyrosine and tyrosinate-tyrosinate singlet energy transfers also occur in homopolypeptides (Longworth et al., 1969). King (1972) has shown that the luminescence properties at 77K of glycyl-tyrosine, tyrosyl-glycine, and tyrosyl-tyrosine are similar to those of tyrosine itselfi In each case the total quantum yield and the fluorescence:phosphorescence ratio are probably close to unity, and the phosphorescence lifetime is 2.6-2.9 s. This behaviour contrasts sharply with that described by Cowgill (1963b) who showed that these and other tyrosine-containing dipeptides had much lower fluorescence quantum yields at room temperature than did tyrosine itself. The tetrapeptide L-cystinyl-bis-L-tyrosine (Fig. 7) has received considerable attention. It has a low fluorescence quantum yield at room temperature (~p = 0.02) (Cowgill, 1967). At 77K the phosphorescence is very feeble indeed (4'P = 0.01), and occurs at an unusually high wavelength (~, = 420 nm), although the phosphorescence lifetime is the same as that of N-acetyl-L-tyrosinamide (Longworth, 1968). The reduction product, L-cysteinyl-Ltyrosine, has a room temperature fluorescence quantum yield intermediate between those of r-cystinyl-bis-L-tyrosine and those of other tyrosine dipeptides (Cowgill, 1967), but at 77K its luminescence yields are equal to those of tyrosine (Churchich and Wampler, 1971). It

Low-TEMPERATURE LUMINESCENCESTUDIESOF PROTEINS AND RELATED MOLECULES

55

thus appears that both the fluorescence and phosphorescence of tyrosine are affected by vicinal sulphur atoms: phosphorescence is reduced more than fluorescence, and disulphide bonds may have a greater effect than sulphydryl groups. These findings are reflected in the properties of oxytocin, a cyclic peptide in which the only aromatic amino acid is a tyrosine residue adjacent to a disulphide linkage (Fig. 7). The fluorescence properties of this peptide are similar to those of free tyrosine, though the quantum yield is reduced by 75 ~o at 298K and 50 ~o at 77K. Again the phosphorescence is unusual, having the low quantum yield and red-shifted spectrum shown by L-cystinyl-bis-Ltyrosine, but the phosphorescence lifetime is normal (Cowgill, 1964; Longworth, 1968). This behaviour, which is apparently typical of systems containing tyrosine residues close to disulphide bridges, may be ascribed in part to a decreased intersystem crossing rate, but the disulphide bond may also affect internal conversion processes. Peptides containing tyrosine and tryptophan are treated separately (see below, section IVD).

Tyr

ys

,,-ArgNH2 ~ G l u

i 1

i

lie

ys

Pro ~

? Leu

~Gly~C--NI'I2

FIG. 7. Structure of bovine oxytocin.

C. Tryptophan and Tryptophan-containing Peptides The absorption spectrum of tryptophan (Fig. 8) has maxima at 220 nm (E = 36,000) and 208 nm (E = 5500). The long-wavelength band which is normally used in luminescence measurements is actually comprised of two electronic transitions, as discussed by Weber (1960) and by Konev (1967): it extends to higher wavelengths than the long-wavelength band of tyrosine, a point of considerable practical value in studies of molecules containing both amino acids (see below, section IVD). The fluorescence maximum of tryptophan in neutral aqueous solution at room temperature is at 350 nm, i.e. the Stokes shift is much larger than that of tyrosine. The difliculties inherent in the determination of absolute quantum yields are nowhere better illustrated than in the case of tryptophan. In numerous studies, little agreement has been achieved, the values obtained ranging from 0.11 to 0.20 (Borrensen, 1967; Bridges and Williams, 1968; Teale and Weber, 1957). There is general agreement, however, that tryptophan fluorescence is enhanced by deprotonation of the amino group at pH 10, but is quenched at still higher pHs and also in acid solution (pH ~< 3). The wavelength of maximum fluorescence of tryptophan (and of the related molecule indole) at room temperature is solvent dependent, being as low as 310 nm for N-acetyl tryptophan methyl ester in n-hexane. There is a general tendency for Ar to increase with the polarity of the solvent. This effect has received much attention, as the tryptophan fluorescence maxima of proteins show similar variations,

56

J.N. MILLER

Tryptophan

200

3OO

SO0

4OO

~ , nn3

FIG. 8. Absorption, fluorescenceand phosphorescence spectra, respectively, of tryptophan in neutral solution. which have been used to deduce the polarity of the environment of the residues concerned. It is unlikely that this effect is due simply to changes in the bulk dielectric constant of the solution, since the addition of low concentrations of polar solvents to non-polar ones produces disproportionately large red-shifts in AF (Van Duuren, 1961). More plausible explanations involve the possible formation of exciplexes (excited state complexes between solute and solvent molecules), or changes in the relative contributions of emissions from the two excited singlet states of tryptophan (see above) (Walker et al., 1967; Kurtin and Song, 1969). An alternative method for studying the environment of tryptophan residues is based on the finding that the ~bF of tryptophan at room temperature is more than doubled by replacing solvent H 2 0 by DzO (Stryer, 1966). This enhancement occurs to different extents in tryptophan-containing molecules, possibly reflecting varying degrees of exposure to solvent of the fluorescent residues. All these solvent effects on fluorescence disappear when measurements are made at 77K, and the corrected AF value for tryptophan at this temperature may be as low as 312-315 nm (Longworth, 1966). High resolution spectra also show shoulders in the fluorescence spectrum at higher and lower wavelengths. The phosphorescence spectrum of tryptophan (Fig. 8) shows several well-resolved maxima at approximately 406, 433 and 456 nm and minor features at higher wavelengths. Tryptophan phosphorescence cannot be detected at wavelengths below 395 nm, and can be distinguished from tyrosine phosphorescence on this basis, and on the basis of lifetime measurements. Numerous re values have been obtained for tryptophan (Longworth, 1971) but the most modern results seem to be close to 6.7 s. There is some evidence that the total luminescence quantum yield of tryptophan at 77K is less than 1. Longworth (1962) obtained the values ~F = 0.64; ~, = 0.16; ~F ÷ ~, = 0.80; ~F/~P = 4.00, and Bishai et al. (1967) obtained ~v ---- 0.72; ~e = 0.17; 5bv q- ~e = 0.89; ~r/'~e = 4.24. In addition, the latter authors detected an increase in total quantum yield in strong acid, largely because of an increase in phosphorescence (~r = 0.74; ~, =

Low-TEMPERATURE LUMINESCENCESYUDI~SOF PROTEINS AND RELATED MOLECULES

57

0.26), and Busel and Burshtein (1970) found that a similar effect occurred in D20. Other values obtained for the ratio ffF/ff~, include 3.85 (Busel and Burshtein, 1970), 3.9 (NagChaudhuri and Augenstein, 1964), and 3.55 (King, 1972). As in the case of tyrosine addition of heavy atoms in the form of KBr or KI reduces this ratio. The phosphorescence lifetime is also reduced, and the decay can no longer be expressed as a single exponential: the latter effect is of uncertain origin, but may reflect the presence of tryptophan molecules in different microenvironments. Whereas incorporation of tryptophan into dipeptides normally causes substantial reductions in fluorescence quantum yields at room temperature (Cowgill, 1963a), such effects are absent at 77K. Weinryb and Steiner (1968) studied a considerable number of tryptophan derivatives at low temperature, and found that they almost all had similar fluorescence: phosphorescence ratios and phosphorescence lifetimes. Although more recent work (L. A. King and J. N. Miller, submitted for publication) has shown that accurate measurements permit N-terminal tryptophan to be distinguished from C-terminal tryptophan in dipeptides, the variations which do occur are much smaller than the range of values encountered in protein studies. Sulphur atoms, however, exert considerable effects on the luminescence of the indole nucleus: bis-indole-3-methylene disulphide has an extremely low fluorescence quantum yield at room temperature (Cowgill, 1967) and is non-luminescent at 77K (King, 1972). Reduction of the disulphide bond does not change these properties. Methionyl tryptophan, however, has no abnormal properties, and it may be that, in this case, the sulphur atom is too far from the indole nucleus to exert any measurable effect (Cowgill, 1970). Poly-L-tryptophan is of interest in having properties not found either in the monomer or in proteins. In particular, its maximum fluorescence wavelength at room temperature is not solvent-dependent, its fluorescence: phosphorescence ratio at 77K is very high (~r/ffe "~ 70) and its phosphorescence spectrum differs from that of poly-DL-tryptophan (Longworth, 1971). The last observation emphasizes the importance of conformational effects in determining the luminescence properties of polytryptophan. As in the case of polytyrosine, fluorescence polarization spectra demonstrate that intertryptophan energy transfer occurs in poly-L-tryptophan (Longworth, 1971).

D. Peptides containing Tyrosine and Tryptophan The luminescence properties of this group of peptides, which provide model systems for the majority of proteins, have been intensively studied. Fluorescence and phosphorescence spectra are normally dominated by contributions from tryptophan residues, but tyrosine luminescence may also occur, and a number of methods are available for detecting it, even when it is of low intensity. Since the fluorescence emission band of tyrosine overlaps the absorption band of tryptophan, tyrosine-to-tryptophan energy transfer at the singlet level is feasible. It has been estimated that such a process might be 50 ~o efficient at a distance of 1.5 nm (Karreman et al., 1958). In the dipeptides tryptophyl-tyrosine and tyrosyl-tryptophan, this energy transfer is apparently 100 ~o efficient, since no tyrosine luminescence is observed, but the excitation spectra of the tryptophan fluorescence and phosphorescence show the expected contribution from the tyrosyl residues (Cassen and Kearns, 1968; King, 1972). Longworth (1971) describes in detail studies of two tyrosine:tryptophan copolymers, with molar ratios 1 : 1 and 4: 1. By the criteria of spectral distribution of fluorescence and phosphorescence, and phosphorescence lifetime, tyrosine luminescence was again absent.

58

J.N. MILLER

Differences between the absorption spectra of tyrosine and tryptophan were utilized in two ways: the emission spectra of the polymers were identical whether they were excited at 295 nm (where tryptophan absorbs but tyrosine does not) or at 278 nm, where both amino acids absorb. This was further proof of the absence of tyrosine emission. Secondly, the quantum yield of the 1 : 1 polymer was independent of the excitation wavelength, but in the 4:1 polymer a wavelength-dependence of ~F was noted. This indicated that the energy transfer was 100~o efficient in the former case, but not in the latter. A complete analysis suggested a transfer efficiency of some 50 ~o, the remainder of the energy absorbed by tyrosine residues being dissipated by internal conversion. In the peptide hormone corticotrophin, which contains 39 amino acids including two tyrosines and one tryptophan, feeble tyrosine fluorescence at room temperature was detected by Eisinger (1969) by differential excitation at 275 and 293 nm. Eisinger also estimated the efficiencies of energy transfer processes in various fragments of the hormone. It is thus evident that tyrosine luminescence may be hard to detect in molecules where tryptophan is also present. If the number and disposition of the residues are appropriate, efficient tyrosine-tryptophan energy transfer may occur: in many cases, however, this mechanism is inadequate for the dissipation of energy absorbed by tyrosine, and the energy loss is due to internal conversion processes. At pHs at which tyrosine residues are ionized, the luminescence properties of molecules containing tyrosine and tryptophan change dramatically. Tryptophan phosphorescence is greatly enhanced, and the fluorescence signal reduced, and it has been suggested that tryptophan-to-tyrosinate energy transfer occurs at the singlet level, and the reverse process at the triplet level (Steiner and Kolinsky, 1968). Triplet-triplet energy transfer, however, is distinguished from the resonance processes which occur at the singlet level by being an electron transfer process: it would thus be expected to be operative over short ranges only, and may be insufficient to explain the above effects. V. PROTEIN LUMINESCENCE A. Class A Proteins

For luminescence studies proteins can be grouped into two classes (Teale, 1960): class A proteins contain tyrosine but not tryptophan, and class B proteins contain both luminescent amino acids. Less than a dozen class A proteins have been studied in detail, although a few others, including aspartate transcarbamylase R, tryptophan synthetase, and E. coli acyl carrier protein, remain uninvestigated. At room temperature the fluorescence maximum of all these proteins (~F ~ 304 nm) seems to be unaffected by the environments of the tyrosine residues and is similar to that of the free amino acid. In most cases, however, the quantum yields of fluorescence of the tyrosine residues are much less than that of tyrosine itself. Values obtained are normally in the range 0.01-0.05 (summarized by Longworth, 1971) although AS-3-ketosteroid isomerase is an apparent exception (Wang et al., 1963). A number of factors may contribute to this effect, including quenching by peptide bonds, carboxylic acids and disulphide linkages (see below), complex formation involving other side-chains, and inter-tyrosine energy transfer, but the relative significance of these factors has not been established in individual cases. At 77K the fluorescence maxima of class A proteins are slightly blue-shifted (~r-~ 298 nm), and the fluorescence quantum yields are increased, though they remain lower than

Low-TEMPERATURE LUMINESCENCE STUDIES OF PROTEINS AND RELATED MOLECULES

59

that of tyrosine. Phosphorescence characteristic of tyrosine has also been observed in a number of cases (Vladimirov and Burshtein, 1960; Douzou et al., 1961) although the lifetime (Tv ~ 2.1 s; Longworth, 1961) may be somewhat lower than that of the free amino acid. Detailed investigations of the low-temperature luminescence of ribonuclease A and insulin have been described. In view of the wealth of structural information obtained from X-ray crystallographic studies of these proteins (Kartha et al., 1967; Adams et al., 1969), this emphasis is not surprising: it is unfortunate, however, in that they appear to be far from typical class A proteins, and systematic work on other members of the class using sensitive, high-resolution equipment is desirable. Ribonuclease A has a very low phosphorescence quantum yield at 77K (~e = 0.05) and the tyrosine phosphorescence lifetime is reduced to 1.4 s (Churchich and Wampler, 1971). In addition the phosphorescence spectrum is shifted to longer wavelengths (Ap = 410 nm; Longworth, 1968) and is similar to the spectra obtained from oxytocin and L-cystinyl-bis-Ltyrosine. It has been suggested that most of the luminescence of RNase A originates in tyr-73 with energy transfer from tyr-76 and tyr-115 possibly contributing (Longworth, 1971). These are the three exposed tyrosine residues in the molecule (there are six altogether) and tyr-73 is close to two disulphide bonds. This hypothesis is supported by the finding that, after reduction (or reduction and carboxymethylation) of the disulphide bonds the fluorescence :phosphorescence ratio (c/,~/4,v = 1.1) and the phosphorescence lifetime (~-p = 2.3 s) of the enzyme are restored to values closer to those of tyrosine itself. The quantum yield of fluorescence of ribonuclease A at 77K is given as 0.2 by Longworth (1971) and 0.09 by Churchich and Wampler (1971). The low-temperature luminescence properties of insulin are also characteristic of tyrosine residues close to disulphide bonds. Again the phosphorescence is very feeble (~e = 0.16, ~be = 0.04, ~F/~be ---- 4.0) and of short lifetime (re = 1.4 s), but abnormalities are removed by reduction and carboxymethylation (fir = 0.25, fie ---- 0.23, ffe/~e = 1.1, re = 2.1 s) (Churchich and Wampler, 1971). Only one tyrosine residue in insulin, tyr-A19, is adjacent to a disulphide linkage and this residue may therefore be the source of most of the luminescence even though another tyrosine, BI6, is more completely exposed (Menendez and Herskovits, 1969). In alkaline solution the low-temperature luminescence of class A proteins is similar to that of tyrosine itself, i.e. they are feebly fluorescent and strongly phosphorescent. In this respect ribonuclease A and insulin behave like other proteins of the class, presumably because denaturation of the molecules has removed the interactions between tyrosine residues and disulphide bonds. These results confirm that vicinal disulphide linkages exert profound effects on the luminescence of tyrosine residues. The major effect may be an enhanced radiationless deactivation of the triplet state accompanied by an increase in the intersystem crossing rate constant--the latter would account for the unusually low ~r values. However, since the percentage reduction in the phosphorescence lifetime is less in some cases than the fall in ~e, it may be that the intersystem crossing rate is decreased and that internal conversion at the singlet level is the major effect. Two other recent observations of interest may be noted. Kimura and Ting (1971) have shown that the class A protein adrenodoxin, which contains two iron atoms and two labile sulphur atoms, has an extraordinarily high fluorescence wavelength at room temperature (AF = 331 nm). The apoprotein shows a normal tyrosine fluorescence (Ar -----304nm). Since the holoprotein and apoprotein have similar properties at 77K (,~e ---- 315 nm) exciplex formation (cf. section IVC) may be involved. Pollet et al. (1972) made the surprising discovery that certain Bence-Jones proteins (immunoglobulin light chains of both K and ;~ p.m 28---¢

60

J.N. MILLER

classes) exhibited mostly tyrosine fluorescence at room temperature, even though tryptophan residues were also present. On heating to 55-65°C, normal tryptophan emission was detected. Similar studies have recently been extended to include low-temperature measurements (Longworth et al., 1973). In view of the intensive investigations devoted to many other aspects of the properties, structures and functions of the immunoglobulins, the native luminescence of these molecules has been relatively little studied. B. Class B Proteins 1. Introduction The great majority of proteins contain tryptophan as well as tyrosine and their luminescence is dominated by emissions from tryptophan residues. Numerous proteins of this class have been studied, but in some cases only room temperature data are available. Where low-temperature measurements have been made, however, they have been shown to be extremely valuable in studies of tyrosine contributions to total luminescence, the environment of tryptophan residues, the effects of sulphur-containing amino acids, and interactions with metal ions and other prosthetic groups. Phosphorescence lifetimes and quantum yield ratios (~r/~e) are particularly useful parameters. 2. Luminescence Properties of Typical Class B Proteins The total luminescence spectrum of a typical class B protein at low temperature bears a superficial resemblance to that of tryptophan itself. In the absence of the solvent-linked effects which cause considerable variations in the fluorescence maxima observed at room temperature, fluorescence maxima at 77K are all very similar, being close to 325 nm in corrected spectra; denaturation processes normally produce no wavelength shifts. In addition, shoulders in the spectrum can frequently be discerned at higher and lower wavelengths. The fluorescence spectra of some proteins, e.g. elastase, ribonuclease T1 (Longworth, 1971), lysozyme, a-lactalbumin (L. A. King and J. N. Miller, submitted for publication), exhibit a much better defined vibrational fine structure; it is assumed this indicates that the fluorescence originates in a single tryptophan residue, or possibly in several tryptophans in similar environments. On the other hand, the fine structure is largely absent from the spectra of some proteins which possess only one tryptophan, e.g. human serum albumin, and is not well-defined in the spectrum of the free amino acid. Tryptophan fluorescence quantum yields at 77K show wide variations (Table 1), comparable with those found in roomtemperature measurements. Most of the values fall below that of tryptophan itself, but a few proteins, notably lactate dehydrogenase and ribonuclease (King, 1972), have high fluorescence quantum yields. The tryptophan phosphorescence of class B proteins also closely resembles that of the free amino acid: the peak maxima in the highly structured spectra show little variation from one protein to another. Phosphorescence quantum yields vary considerably (Table 1), but are less than that of tryptophan. Some quenching at the triplet level is also suggested by the finding that tryptophan phosphorescence lifetimes in proteins are normally rather lower than that of tryptophan itself: again considerable variations occur. In the single protein studied in detail, however, the major cause of energy loss at 77K in ~-chymotrypsin was found to be internal conversion at the singlet level (Galley and Stryer, 1969). Table 1 shows that values of the fluorescence:phosphorescence ratio ~e/~, are mostly in the range 5.0 ! 1.5: the value for tryptophan is probably close to 4 (see above, section IVC).

Low-TEMPERATURE LUMINESCENCESTUDIES OF PROTEINS AND RELATED MOLECULES

61

TABLE I. TRYPTOPHAN LUMINESCENCE PROPERTIES OF TYPICAL CLASS B PROTEINS AT 77K

"rp

~P

Protein Human serum albumin Ribonuclease T2 a-Chymotrypsin Pepsin Papain fl-Laetoglobulin Deoxyribonuclease

0.11 0.70 0.18 0.47 0.50 0.69 0.80

0.03 0.15 0.03 0.11 0.07 0.14 0.14

3.7 4.7 6.0 4.3 7.1 4.9 5.7

(s)

References

6.0 6.0 5.9 5.9 5.2 5.0 5.9

1

1 1,2 3 1 3 3

1 Longworth(1971), and referencestherein. 2 Konev (1967), and referencestherein. a King (1972). 3. Tyrosine Luminescence in Class B Proteins The contributions of tyrosine residues to the total luminescence of class B proteins have been the subject of considerable controversy. The authors of several early publications concluded that tyrosine luminescence could not be observed if tryptophan residues were also present. Nor was there any evidence to suggest that tyrosine-tryptophan energy transfer could explain this. In an extreme case Teale (1960) detected only tryptophan fluorescence in human serum albumin, in spite of the great preponderance of tyrosine in this protein. Later, however, a number of workers (Weber, 1961, Longworth, 1961 ; Teale 1961) detected small tyrosine emission bands in the spectra of class B proteins, and the modern view is that the total absence of tyrosine luminescence is rare. Longworth (1968) could find no tyrosine emission in studies of ribonuclease/'i, but the contrary view was taken by Pongs (1970). The apparently irreconcilable results often encountered in this field reflect the considerable experimental problems involved in detecting tyrosine luminescence when tryptophan is present, especially in experiments at room temperature. The tyrosine contribution does not normally exceed 10-15 % of the total luminescence and may be much less than this (see below); the Stokes shift of tyrosine fluorescence is small, so scattered light signals may interfere; and fluorimeters vary widely in their sensitivity and resolution. Nevertheless, Longworth's claim (1971) that tyrosine fluorescence can only be observed on especially constructed instruments of high sensitivity and resolution may be an exaggeration. Several indirect methods have proved suitable for detecting tyrosine fluorescence. The matrix procedure of Weber (1961) and the difference spectrum method both rely on the finding (see above, section IVD) that both tyrosine and tryptophan can be excited at 278 nm but that tyrosine absorption is negligible at 295 nm. In the difference method, fluorescence spectra excited at these two wavelengths are normalized at 370 nm (where only tryptophan fluorescence is significant), and the spectrum excited at 295 nm subtracted from that obtained at 278 nm. The resulting difference spectrum reveals any tyrosine fluorescence. Longworth (1971) has discussed this method in detail. Alternative approaches to the problem eliminate the luminescence of tryptophan residues either by oxidation with Nbromosuccinimide or by promoting energy transfer from tryptophans to bound dye molecules: only tyrosine fluorescence then remains (Teale, 1961; Chen and Cohen, 1966; Churchich, 1965). The value of such methods naturally rests on the assumption that the proteins are not denatured or otherwise affected by the modification procedures.

62

J.N. MILLER

Low-temperature luminescence measurements, with a phosphoroscope included in the optical arrangement to eliminate scattered light and fluorescence, provide much the best method of detecting tyrosine luminescence. Not only are the phosphorescence lifetimes of tyrosine and tryptophan quite different, but the phosphorescence maximum of tyrosine normally occurs at a wavelength (385-390 rim) at which tryptophan does not phosphoresce at all. Any phosphorescence signal with a lifetime of ~ 2 s which occurs at wavelengths below 395 nm can thus be attributed to tyrosine. Of course, the difference spectrum technique can also be applied at 77K to evaluate tyrosine luminescence contributions. The only complications in these methods would appear to be the occurrence of background luminescence of the solvent, and the possibility that certain tyrosine residues may be phosphorescent but not fluorescent (Steiner, 1971). In alkaline solution the low-temperature spectra of class B proteins are dominated by contributions from tyrosinate ions (Truong et al., 1967) whose phosphorescence properties in particular are readily distinguished from those of tryptophan. Since many proteins are denatured in alkali, however, it would be unwise to deduce from observations of tyrosinate spectra that tyrosine residues necessarily contribute to luminescence in the native state. Application of this range of methods shows that the fluorescence and phosphorescence spectra, and phosphorescence lifetimes, of tyrosine residues in class B proteins are very similar to those found in other tyrosine-containing molecules. Phosphorescence excitation spectra have also been obtained and a blue shift, comparable to that encountered in absorption spectroscopy, has been observed in acid solutions (Longworth, 1961). Unusual phosphorescence emission spectra, such as those of oxytocin, insulin, and RNase A, have apparently not been discovered. Tyrosine fluorescence quantum yields in class B proteins at room temperature are generally less than 0.05 (Weber, 1961; Teale, 1961), although higher values prevail after denaturation. At low temperature ~F may be as high as 0.2, indicating that some dynamic quenching effects may be abolished. Since there is little evidence for general enhancements of the fluorescence of tryptophan residues, it may be easier to distinguish directly tyrosine contributions to fluorescence bands at 77K than at room temperature. Phosphorescence quantum yields are low (< 0.05) but are also increased on denaturation. The dominance of tryptophan luminescence in class B proteins is easily understood in terms of these low tyrosine quantum yields, which are caused, as discussed in section VA by a variety of dynamic and static quenching effects. The possibility that tyrosine-tryptophan energy transfer further reduces tyrosine luminescence must also be considered. Such energy transfers have generally been studied by considering the dependence of the quantum yield of tryptophan fluorescence on the exciting wavelength (cf. section IVD). The extent to which energy transfer processes occur will naturally vary with the numbers, proximity and orientations of the tyrosine and tryptophan residues in the proteins. Thus in some proteins energy transfer is negligible, while in others, notably papain (Weinryb and Steiner, 1970), it is considerable. In most cases, however, it is only one of a number of factors contributing to the low tyrosine quantum yields. The role of tyrosine residues in the luminescence of typical class B proteins can thus be summarized as follows. Although the fractional light absorption due to tyrosine varies from protein to protein according to the amino acid composition, most of this energy is dissipated via non-luminescent pathways. Some may be transferred to tryptophan residues, leaving at most a small fraction to be re-emitted from the tyrosine residues themselves. Some representative data are given in Table 2.

63

Low-TEMPERATURE LUMINESCENCESTUDIES OF PROTEINS AND RELATED MOLECULES TABLE 2, TYRO$INE CONTRIBUTIONSTO THE LUMINESCENCEOF CLASS B PROTEINS

Protein Human serum albumin a-Chymotrypsin

Tyr: Trp ratio 17

Fractional tyrosine absorption 280 nm

Tyr ~ Trp energy transfer efficiency

0.85

0.23

Luminescence properties

Refs.

Tyr contributes 25 ~o of phosphor-

1

escence

0.5

0.12

0

Pepsin Papain

3.4 3.8

0.48 0.51

0.25 0.56

~-Lactalbumin Dvalbumin

1 3

0.19 0.34

0.12 0.38

Tyr contributes 10~ of phosphorescence Small tyr luminescence Tyr contributes 15 Yo of phosphorescence Negligible tyr luminescence Tyr contributes 10yo of phosphorescence

1

2 3 4, 5 1

x Longworth (1971), and references therein. 2 Kronman and Holmes (1971). 3 Steiner (1971). 4 Kronman (1967). 5 L. A. King and J. N. Miller, submitted for publication. A major exception to this general pattern is the bacterial protease subtilisin Carlsberg, whose phosphorescence band includes a large and easily recognized tyrosine component (Longworth, 1971), with a normal lifetime (rp = 2.2 s; King, 1972). Horseradish peroxidase (King, 1972), staphylococcal endonuclease (Longworth, 1968) and hemocyanin (Shaklai and Daniel, 1972) also exhibit an unusual degree of tyrosine luminescence. Of these proteins, subtilisin Carlsberg and staphylococcal endonuclease both have high tyrosine :tryptophan ratios (13 and 7 respectively) and fractional tyrosine absorption (0.8 and 0.7 respectively). Also, both lack disulphide bonds, which may be important quenchers of tyrosine luminescence. Hemocyanin, on the other hand, has a lower tyrosine:tryptophan ratio (~2.5) and does contain disulphide linkages.

4. Class B Proteins showing Unusual Tryptophan Luminescence Several class B proteins have been studied whose luminescence properties do not conform to the normal pattern outlined in section VB, 1 above. The best characterized of these is lysozyme, normally isolated from hen egg-whites. This protein would in any event be of interest, as X-ray studies have thrown much light on its structure, and the low tyrosine: tryptophan ratio (3 and 6 residues respectively) facilitate observations of tryptophan luminescence. In practice, tyrosine luminescence is negligible, and tyrosine-tryptophan energy transfer is also unimportant (Imoto et al., 1971). Churchich (1964, 1966) showed that the fluorescence quantum yield of lysozyme is low, but the most unusual luminescence properties are observed at low temperature. Figure 9 compares the total luminescence spectrum of lysozyme with that of a typical class B protein: the fluorescence:phosphorescence ratio is evidently much higher than usual (it may be as high as 50 according to Longworth, 1966), the fluorescence spectrum exhibits considerable vibrational fine structure, and the vibrational peak maxima in the phosphorescence band are at unusual wavelengths. The phosphorescence decay of lysozyme is also abnormal (Churchich, 1966). Very short-lived components (Te ~ 1 s) apparently account for most of

64

J . N . MILLER

the phosphorescence, but there is a further component whose lifetime (re ~ 4 s), while longer, is still less than that of most class B proteins. It seems certain that this unusual behaviour reflects the proximity of tryptophan residues to disulphide bonds: in all, five such residues are close to disulphide linkages in the native conformation of lysozyme. Studies on lysozyme derivatives prepared by oxidation with iodine and with N-bromosuccinimide suggest that two of these tryptophans, at positions 62 and 108, are responsible for most of the fluorescence of the enzyme at room temperature (Imoto et al., 1971). Furthermore, denaturation of the enzyme with guanidine hydrochloride has little effect on the fluorescence quantum yield, presumably because the tryptophan residues remain close to the disulphide bonds (Kronman and Holmes, 1971). After I A :

lysozyme

B : typical

200

1

class B protein

300

4O0

SO0

~,nm

FIG. 9. Total luminescence spectrum of lysozyme at pH 8.5, compared with that of a typical class B protein.

reduction of these bonds, the phosphorescence decay of lysozyme can be expressed as a single exponential (rp = 4 s), although the phosphorescence remains feeble (Churchich, 1964). Much interest has been aroused by the finding of Browne et al. (1969) that the primary sequence of the whey protein ~-lactalbumin could be folded into the backbone conformation of lysozyme: many similarities between the two proteins have been noted (reviewed by Lyster, 1972). Bovine ~-lactalbumin has only four tryptophan residues (again close to disulphide bonds) and four tyrosine residues. Studies of its fluorescence at room temperature show a number of similarities with the properties of lysozyme, including a low quantum yield and negligible tyrosine contribution (Kronman, 1967; Kronman and Holmes, 1971). Unlike lysozyme, however, its fluorescence quantum yield is much enhanced on denaturation, even when the disulphide bridges remain intact. Low-temperature studies (L. A. King and J. N. Miller, to be published) confirm that a-lactalbumin luminescence is generally similar to that of lysozyme, although certain differences are apparent. Preliminary studies by King (1972) on various immunoglobulin fractions indicate that these proteins also have unusually high fluorescence:phosphorescence ratios at 77K. It may be significant that Litman et al. (1970) have shown that tryptophan residues in immunoglobulin light chains are often close to cystine residues. Phosphorescence lifetimes, however,

LoW-TEMPERATURE LUMINESCENCESTUDIESOF PROTEINS AND RELATED MOLECULES

65

were generally not much lower than usual, tending to confirm (cf. the case of lysozyme above) that abnormal lifetimes and abnormal fluorescence:phosphorescence ratios may reflect different structural features. Purkey and Galley 0970) were able to resolve the phosphorescence spectrum of horse liver alcohol dehydrogenase into two components separated by ~ 300 cm- 1. Evidence was presented that these components reflected the different environments of the two tryptophan residues present in each subunit. Similar phenomena were observed in the cases of papain, trypsin and yeast alcohol dehydrogenase, but such findings have apparently not been reported by other workers. The role of the Zn 2e ions in alcohol dehydrogenase luminescence has not been investigated, but a number of other metalloproteins exhibit unusual properties. Amongst haem proteins, cytochrome c and haemoglobin are non-luminescent, presumably because of energy transfer to the iron-porphyrin system (Weber and Teale, 1959). L-Lactate dehydrogenase isolated from yeast (cytochrome b2), on the other hand, is strongly luminescent. Risler has shown that the fluorescence of this enzyme is partially quenched by the presence of the flavin mononucleotide (FMN) prosthetic group, but not by the haem moieties, and that the reverse is true of the phosphorescence. Calculations suggested that an orthogoral orientation of the F M N and haem groups might account for these effects. The luminescence of two copper-containing proteins has recently been studied. Azurin, isolated from Pseudomonas fluorescens, has an unusual room-temperature fluorescence, with the tryptophan maximum at the low wavelength of 308 rim. Tyrosine contributions are absent and the fluorescence quantum yield is tripled by removal of the single copper atom (Finazzi-Agro et al., 1970). Low-temperature measurements showed that Cu 2~ also reduced the phosphorescence by two-thirds without, however, affecting its lifetime (¢p -----5.8 s). Singlet internal conversion was proposed as the quenching mechanism, and the effects of Hg 2~ and Ag e ions were also studied (Finazzi-Agro et al., 1973). Shaklai and Daniel 0972) studied the phosphorescence of hemocyanin isolated from Levantina hierosolinia. A considerable tyrosine contribution was detected and was enhanced in the presence of copper, while tryptophan phosphorescence was relatively unaffected. Oxygenation of the protein quenched both tyrosine and tryptophan luminescence, the copper-oxygen complexes acting "sinks" in energy transfer processes. VI. C O N C L U S I O N S

Low-temperature luminescence measurements are clearly capable of supplying much detailed information on the static structural features of proteins and related molecules, on the photochemical processes that follow the absorption of light by these molecules, and on their structural transitions such as denaturation. As experimental techniques improve and become more widely available, it may be anticipated that they will be applied to the study of many more proteins, especially those containing prosthetic groups, including metal ions. The availability of a sampling system in which organic solvents are not required will be of particular value in reducing background luminescence, and in studying pH effects. Indeed it may soon become routine practice to study protein luminescence at 77K as well as at room temperature. VII. ACKNOWLEDGEMENTS

Luminescence studies in this laboratory could not have been developed without the support and encouragement of Prof. R. F. Phillips and Dr. D. Thorburn Burns. I have also

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J . N . MILLER

had many stimulating conversations with Dr. J. W. Bridges, of the Department ot' Biochemistry at the University of Surrey, and with Dr. L. A. King, now at the Home Office Central Research Establishment, Aldermaston.

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