Raman spectroscopic studies of some biochemically relevant molecules

VIBRATIONAL SPE OPY ELSEVIER

Vibrational Spectroscopy 9 (1995) 111-120

Raman spectroscopic studies of some biochemically relevant molecules S.E. May Colaianni

a,

j. Aubard b, S. H¢ime Hansen c, O. Faurskov Nielsen

a, *

a Chemical Institute, H.C. Orsted Institute, Copenhagen University, Universitetsparken 5, DK-21 O0 Copenhagen, Denmark b ITODYS, Universit~ Paris VII, 1, rue Guy de la Brosse, F-75005 Paris, France e Novo N o r d i s k A / S , Biopharraaceuticals Division, DK-2820 Gentofte, Denmark Received 4 June 1994

Abstract Near-infrared (NIR) Raman spectra of the protein aprotinin, in both powder form and aqueous solutions, are presented. The amide I and amide III bands give information about the secondary structure. The conformation around the sulphur bridges and the environment of tyrosine were also studied. Due to the low scattering efficiency, only aqueous solutions in the concentration range 2-20% (w/w) were used. Use of a windowless cell improved the quality of the spectra, as compared to spectra obtained with quartz cells. Fluorescence can be a serious problem in Raman studies of biologically relevant molecules. Some examples are shown, which illustrate that the use of NIR excitation can frequently eliminate this fluorescence. Heating effects give rise to serious problems with excitation at 1064 nm in the NIR-FT-Raman spectrum of some strongly coloured macromolecules, like haemoglobin. In order to avoid complications due to both heating and fluorescence, an excitation wavelength around 800 nm is suggested. A preliminary surface enhanced Raman (SER) spectrum of a peptide nucleic acid (PNA) in aqueous silver colloid solution is shown. Low-frequency Raman spectra of aprotinin in aqueous solution are presented. The low-frequency limit in the NIR-FF-Raman spectrum is ~ 80 cm-1. Several models are used to describe the bands assigned to hydrogen bonding in the systems. The low-frequency modes can be of importance for the formation and breaking of hydrogen bonds, and thus may be of importance for biological activity. Keywords: Aprotinin; Proteins; Raman spectrometry, near-infrared; Raman spectrometry, surface enhanced; Secondary structure

1. Introduction Raman spectroscopy has become a valuable tool for studying molecules of biological interest [1-3]. This paper shows how Raman spectroscopy can be used in conformational studies of aprotinin, a protein with 58 amino acid residues. This protein is a synthetic form of the basic pancreatic trypsin inhibitor (BPTI). The Raman spectra of a natural bovine pro* Corresponding author. 1 Registered trademark of Bayer Leverkusen, Germany.

tein, Trasylol x, was published back in 1975 [4]. This spectrum was obtained by excitation in the visible (Vis) region at 488 nm. The spectra all showed some background features, and thus it seemed worthwhile to reinvestigate this protein using excitation in the NIR. The main conclusion in the previous paper on natural BPTI [4] was that differences in the main chain conformation of BPTI occurred between the solid state and aqueous solutions. This statement is not in accordance with a number of conformational studies of BPTI using other methods [5-8]. And according to our results, which also include solutions in

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deuterium oxide, the Raman spectra confirm that no drastic conformational changes occur in going from the synthetic form of BPTI, aprotinin, in the solid state to aqueous solutions. It is well-known that Vis-Raman spectra of solid proteins and their aqueous solutions often show undesirable backgrounds, due to either fluorescent parts of the macromolecules or to unavoidable impurities. Using excitation in the NIR region can alleviate this problem for many biological samples. However, one drawback with using NIR excitation is that heating of some coloured solid proteins can occur and produce large backgrounds. This problem is illustrated in some detail by NIR-Fr-Raman spectra of haemoglobin. The advantage of a windowless cell is shown using an aqueous solution of lysozyme. One disadvantage of using Raman spectroscopy to study aqueous solutions of biological molecules is that very concentrated solutions must be used, since the sensitivity of the method is not high. However, resonance Raman techniques can be used to study aqueous solutions at very low concentrations, which may be of physiological relevance. The present contribution does not describe real resonance Raman spectroscopy (RRS), but a preliminary result obtained with surface enhanced Raman spectroscopy (SERS) will be shown. This technique is useful in general studies of aqueous solutions at low concentrations [9], as illustrated with SER spectra of an aqueous solution of a peptide nucleic acid, PNA, at low concentrations. The low-frequency region of the vibrational spectrum, from approximately 10 to 300 cm -1, is very important for studying intermolecular interactions. In this part of the spectrum, the bands observed may be assigned to modes which involve atoms in hydrogen bonds [10-13]. The low-frequency spectrum of aprotinin, in both solid state and aqueous solution, will be presented and related to different theoretical models.

2. Experimental 2.1. Chemicals

Human haemoglobin and lysozyme from chicken egg white were purchased commercially. The lyophilized aprotinin sample was obtained from Novo

Nordisk A / S , Biopharmaceuticals Division, Gentorte, Denmark (batch no. A38H32). The protein solutions were made using bidistilled water or deuterium oxide, and no buffer, and were generally used within 5 h after preparation. The PNA was a gift from Copenhagen University [14]. 2.2. N I R - F T - R a m a n

The instrument used was a Bruker IFS-66 interferometer with a FRA-106 Raman module and a cooled Ge-diode detector. The excitation source was a Nd 3+-YAG laser (1064 nm) in the backscattering (180 °) configuration. The focused laser beam diameter was ~ 100/zm and the spectral resolution was 6 cm-1. At the sample, the laser power was approximately 300 mW, and the total number of scans for each spectrum was approximately 10 000, except for the PNA spectrum, which consisted of 4000 scans. The NIR-FT-Raman spectra were obtained in the frequency range from ~ 80 to 3500 cm -1. We have corrected all the NIR-FT-Raman spectra shown here for the instrumental response as follows. It is assumed that an incandescent lamp is a black body emitter at a temperature of 3200 K. The choice of this temperature is somewhat arbitrary. The emission spectrum of the lamp was obtained and was divided by a black body radiation spectrum calculated at 3200 K using the expression Era d --

2 × 1 0 7 1 r h c ~ a / [ e x p ( l O O h c ~ , / k a T ) - 1]

(W/mZcm -1)

(1)

The measured NIR-FT-Raman spectra were then divided by this resultant spectrum, giving the corresponding instrumentally corrected spectrum. The correction of the spectra are in particular important in the region above 3000 cm-1, where the detector sensitivity drops rapidly off and in the low-frequency region due to the effects of filters. A narrow band at ~ 85 era-1 is observed in all the NIR-Fr-Raman spectra, which is due to a laser emission line, and we eliminated this line from all the NIR-Fr-Raman spectra as follows. We obtained the spectrum of the exciting laser light reflected back from a mirror. This gave the laser emission line band shape, which we subtracted from all the measured NIR-Fr-Raman spectra.

S.E. May Colaianni et al. / Vibrational Spectroscopy 9 (1995) 111-120

Spectra of the aqueous solutions were obtained by use of a windowless cell, which, in its simplest form as we used, consists of a small hole in a metal block [15].

113

I

A

2.3. Vis-Raman The instrument used was a Dilor Z-24 with a triple monochromator and cooled photomultiplier tube. The excitation source used was an Ar-ion laser (514.5 nm) and the 90 ° scattering configuration was used. The laser power at the sample was approximately 400 mW and the spectral resolution was 2.8 cm -1.

2.4. SERS The SER spectrum was obtained on a DILOR XY spectrometer, and the sample solution contained 0.6 mg of PNA in 0.7 ml of an aqueous silver colloid solution, prepared as previously described [16].

3. Results and discussion

3.1. Use of a windowless cell and conformation studies of aprotinin (BPTI) BPTI has been intensively studied by a number of different methods, and its structure, chemical, physical and biochemical properties have been described in detail [7,17-21]. It contains 58 amino acid residues, three disulfide bridges and four tyrosine residues. We have obtained NIR-Fr-Raman spectra of aprotinin in solid powder form, and in 2, 5, 10 and 20% ( w / w ) H 2 0 and D20 solutions in a windowless cell. Due to the use of pinholes instead of slits, the most efficient scattering geometry in NIR-FT-Raman spectroscopy is the backscattering (180 °) configuration. However, with this configuration the spectrum of the walls of the cells used cannot be avoided. In our experiments the cells used were quartz tubes designed for ESR spectroscopy. This can be a serious problem for weak scatterers like proteins in aqueous solutions. Due to the lower scattering efficiency for NIR radiation as compared to Vis radiation, we used long measurement periods of about 4 h. During this period, some evaporation of the solvent occurred, so the windowless cell was refilled several times during a single experiment. In Fig. 1 are shown NIR-Fl'-Raman

3500

3000

2500

2000 1500 I000 Wovenumbers (cm-I)

500

Fig. l. NIR-FT-Ramanspectra of a 20% (w/w) lysozymeaqueous solutionin (A) a windowlesscell and (B) a quartz tube. spectra of a 20% ( w / w ) aqueous lysozyme solution in (A) a windowless cell (10000 scans) and (B) a quartz tube (6000 scans). We also obtained a Vis-Raman spectrum of 20% ( w / w ) aqueous solutions of aprotinin and lysozyme in the frequency range from 10 to 4000 cm-1. The Vis-Raman spectrum of aprotinin was nearly identical to the spectrum obtained by NIR excitation, although the relative intensities were slightly different, because the Vis-Raman spectrum was not corrected for the spectrometer response. However, no broad background seemed to appear, as observed in the older Raman work using 488 nm excitation [4]. Thus, the synthetic form of BPTI, aprotinin, could have been investigated with Vis excitation. The backgrounds appearing in the previous investigation of natural BPTI, Trasylol, is quite often seen in biochemical compounds isolated from living systems, as observed for our lysozyme solution obtained with Ar-ion laser excitation, where a strong, broad fluorescence band made the Raman bands of lysozyme barely detectable. With the NIR-FT-Raman spectrum of the lysozyme solution obtained in the windowless cell, we have obtained a signal-tonoise ratio comparable to that of Lord and Yu [22] in their classic study on concentrated aqueous solutions of lysozyme using visible excitation at 632.8 nm. In Fig. 2 are shown NIR-FT-Raman spectra of (A) aprotinin powder, (B) a 20% ( w / w ) aprotinin deuterium oxide solution in a windowless cell, and aprotinin aqueous solutions of (C) 20% (w/w), (D) 10% (w/w), (E) 5% ( w / w ) and (F) 2% (w/w), all in a

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g C r~

B

~ E~N.... 560 5~0 520 5OO 48O g00 880 060 040 820 3500

3000

Z500

2000

1500

1000

500

Wovenumbers (cm-1)

Fig. 2. NIR-FF-Ramanspectra of (A) aprotinin powder, (B) a 20% (w/w) deuterium oxide solution in a windowless cell, and aqueous aprotinin solutions of (C) 20, (D) 10, (E) 5 and (F) 2% (w/w) in a windowless cell; pH 4.3-4.6. windowless cell. No relative intensity changes seem to occur as a function of the concentration down to 10%. At lower concentrations as shown in Fig. 2E and F the bands are too weak to compare the relative intensities. The secondary structure of biological molecules can be determined by investigating the bands associated with the amide group in the amide I (1620-1700 cm -1) and amide III (1220-1310 cm -1) regions of the Raman spectrum [1,2], because these amide bands show high intensities in the Raman spectra. Fig. 3A I

f

I

/'~

e=

1680

1660 16(`0 16201320 1300 1280 t260 12(,0 1220 Wovenumbers(cm-~)

Fig. 3. The amide I region of NIR-FT-Ramanspectra of (A) aprotinin powder and (]3) a 20% (w/w) deuterium oxide solution in a windowless cell, and the amide III region of NIR-FT-Ramanspectra of (C) aprotinin powder and (D) a 20% (w/w) aqueous solution in a windowless cell. Bands characteristic of specific secondary amide structures are marked with arrows.

Wovenumbers (cmq)

Fig. 4. (A) and (C) show NIR-FT-Raman spectra of aprotinin powder, and (B) and (D) show a 20% (w/w) aprotinin aqueous solution in a windowless cell. The band in A and B is characteristic of the sulfur bridge conformationsand the doublet around 800 cm-1 to the hydrogen bonding of tyrosine. and C indicate that the aprotinin powder has primarily/3-sheet structure ( ~ 1665 cm -1 and 1240 cm -1) and also some a-helical content (1270 cm-1). The amide I band around 1650 c m - 1 arising from an c~helix content may easily be hidden in the broad band observed for the amide I vibrations. A contribution around 1660 cm -1 from a chain belonging to the random coil conformation may also easily be hidden by the other bands. Three different crystal structures have been reported [7,18-20]. The main chain conformation of all three forms are very similar. Differences occur in the helical parts, and especially the degree of order near the terminals is different for the three forms. Fig. 3B and D, in D 2 0 and H 2 0 solutions respectively, indicate that aprotinin has a higher content of random coil and helical structures in aqueous solution. However, this might also reflect a different degree of order, as just mentioned. The amide I band is not useful in conformational studies of aqueous solutions, because the water bending vibration occurs in this region. For this reason, heavy water is used as the solvent in this region (Fig. 3B). The amide III region from 1220 to 1310 c r n - 1 is not useful in studies of heavy water solutions, because the N - H exchange by deuterium changes the vibrational mode for the amide III band, and only the amide III region for the solution of ordinary water is shown in Fig. 3D. By examining the relative heights of the bands at approximately 510, 525 and 540 cm -1, the confor-

S.E. May Colaianni et al. / Vibrational Spectroscopy 9 (1995) 111-120

115

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.03

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o

[3

r~

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7.5

iml

2.$

3500

2.80O

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1400

700

0

Wovenumber (cm-~ Fig. 5. N]R-FT-RamaQ spectra of solid haemoglobin obtained using various power ]eve|s. From bottom to top: 20, 40, 60, 80 and 105 roW. (A) shows the measured spectra and (B) shows the same spectra corrected for the instrumental response.

mations of the molecules in the three disulfide bridges can be inferred [1]. As shown in Fig. 4A and B, in the powder and aqueous solution spectra of aprotinin respectively, the band at ~ 510 cm -1 is consistently the most intense band in the region 500-560 cm -1. This indicates that in the solid state and in solutions of both water and heavy water, the conformation of all three disulfide bridges is the gauche-gauchegauche conformation. A further calculation of the vibrational frequencies of the disulphide groups in BPTI was recently performed [23]. It is possible to study the environments of the four tyrosine side chains in the protein, by examining the

relative heights of the bands at approximately 830 and 856 cm -1 . According to Tu [1], the mole fractions of buried and exposed tyrosine residues, Nburied and N~posed, can be calculated according to the equations gburied + gexpose d = 1

(2)

0.5Nb,ried + 1.25Nexposed = 185o/183 o

(3)

Using these equations, the largest ratio possible for 18so/183 o is 1.25. However, in both the powder (Fig.

4C) and aqueous solution (Fig. 4D) spectra of aprotinin, we have found by curve-fitting analyses, that the area of band at ~ 857 cm-1 is more than 1.25

116

S.E. May Colaianni et al. /Vibrational Spectroscopy 9 (1995) 111-120 1.8.

1.35

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Fig. 6. Calculated emission spectra from an absolute black body. From bottom to top: at 300, 500, 750 and 1000 K, using Eq. 1.

times that of the band at ~ 830 cm-1. According to Eqs. 2 and 3, this would indicate that the tyrosine side chains in aprotinin in both solid state and aqueous solution are all exposed. However, as already pointed out in the previous Raman study of aprotinin [4], the x-ray structure shows that two of the tyrosines are most probably to be considered as buried, thus questioning the quantitative use of Eqs. 2 and 3 for BPTI. The present results obtained by Raman spectroscopy confirm other studies regarding the conformation of aprotinin [24], which point out that the conformation of the aprotinin average solution conformation closely matches the conformation of its crystalline structure. This is because hydrophobic clusters form stability domains in the interior of the inhibitor, which function as pillars for the architecture of the protein molecule [24]. However, the amide I and III bands indicate that smaller differences, mainly due to the helical content, might be present between the solid state and the aqueous solutions. This result is supported by recent measurements by nuclear Overhauser effect (NOE) spectroscopy [25].

3.2. Fluorescence and heating effects Although fluorescence can many times be avoided using NIR excitation, a potential problem is the heat-

ing of solid samples. This is illustrated in Fig. 5, which shows NIR-FT-Raman spectra of solid haemoglobin powder obtained at various incident power levels, with laser excitation at 1064 nm. With increasing incident power, an increasing background is obtained, due to increasing thermal emission. The calculated emission spectra from an absolute black body at 300, 500, 750 and 1000 K are shown in Fig. 6. The energy ( W / m 2) per wavenumber (cm -1 ) was calculated using the expression for the black body radiation spectrum given in Eq. 1. The Ge-diode detector sensitivity decreases rapidly at Raman shifts above approximately 3000 cm -1, and so the measured haemoglobin spectra in Fig. 5A show essentially a black body radiation curve overlapped with the spectrometer instrumental response. The spectra in Fig. 5B are corrected for the instrumental response. The bands just below 3000 era-1 are assigned to aliphatic stretching vibrations. The other bands observed are very broad and no real assignments can be made. At temperatures below approximately 1000 K, heating by the exciting laser source becomes more and more important for exciting lines moving towards the IR region (longer wavelengths). However, the fluorescence problem in biological polymers from unavoidable impurities is expected to increase mov-

117

S.E. May Colaianni et al. / Vibrational Spectroscopy 9 (1995) 111-120 I

I

on

I

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bands in this region can be assigned to modes involving atoms in hydrogen bonds. The low-frequency spectra can be conveniently studied in the R(~) representation [29] R ( ~ ) ot (~L

--

~ ) - 4 ~ [ 1 -- exp(-hvc/kBT)] I(-P)

(4) I

1500

1000 Wovenumbers (cm-1}

500

Fig. 7. Vis-SERspectrumof a peptidenucleicacid H-T10-Lys-NH2 in an aqueous silver colloid solution. ing towards shorter wavelength excitation. Thus, using excitation wavelength around 800 nm, as proposed by Hendra et al. [26], should be optimal for avoiding heating, although fluorescence is a problem in some cases. Recently we have used NIR-FT-Raman to monitor the stepwise solid state Merrifield synthesis of some polypeptides [27]. Investigation by visible excitation was impossible due to the presence of a large fluorescence background.

3.3. SERS studies of PNA In studies of biomolecules, usually only very low concentrations in aqueous solution are of physiological relevance. Because the sensitivity of Raman spectroscopy is low, it can be difficult to study solutions of very low concentrations. Fig. 7 shows a Vis-SER spectrum obtained from an aqueous silver colloid solution of the H-Tm-Lys-NH 2 PNA. PNA is a nucleic acid analogue [14]. The enhanced bands in the SER spectrum are generally assigned to vibrational modes occurring in the part of the molecule which bonds to the silver colloid [28]. The strong band at 791 cm-1 is certainly a ring mode from the base part of the molecule, and bands in the amide I and III regions might be assigned to modes from the pseudo peptide. PNA can bind to DNA through base pairing, and we hope to study this interaction with the SERS technique.

3.4. Low-frequency studies Studies of the low-frequency region of the Raman spectra of biomolecules are very interesting, because

where ~ is the Raman shift, ~ L is the laser frequency, and I(~) is the intensity of the measured Raman spectrum. The central Rayleigh line has a Lorentzian shape, due to pure reorientational relaxations, and so the advantage of using the R(~) representation is that the Rayleigh line is converted into a plateau [30]. This greatly simplifies the investigation of Raman bands of low intensity in the Rayleigh wing. The R(~) representation is an example of the so-called reduced Raman intensities [29,31]. Other approximations can be used, as thoroughly discussed in a recent review by one of us [31]. Fig. 8A shows a measured NIR-FT-Raman spectrum of a 20% ( w / w ) aqueous solution of aprotinin, and in Fig. 8B the laser emission line has been subtracted. Fig. 8C shows this spectrum instrumentally corrected, and Fig. 8D shows its R(~) representation. Three different models were used to describe the low-frequency band at approximately 100 cm -1, which appears in the spectra of all the samples studied here. The first model used describes these lowfrequency bands in terms of the density of states of

A i g

i

i

~

B c

o~

350

300

250 200 150 Wovenumbers (cmq)

I00

50

Fig. 8. NIR-FF-Ramanspectraof a 20% (w/w) aprotininsolution in a windowlesscell. (A) shows the measuredspectrum,(B) shows the laser emission line subtracted, (C) shows the instrumentally correctedspectrumand (D) shows the R(~) representation.

S.E. May Colaianniet al./ VibrationalSpectroscopy9 (1995)111-120

118

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300

~0

B

el

,sI ..::

// /" e

/ sS

:

/ ,,,"

ssI.... ."

300

200

1013

100

Wovenumbers (cm-~) Fig. 9. The solid lines show the R(~) representation of NIR-FFRaman spectra (full line) of (A) aprotinin powder and (B) a 20% (w/w) aprotinin aqueous solution in a windowless cell, along with their best-fit damped oscillator model curves (dashed lines) and Gaussian cage model curves (dotted lines). In B the dashed and full lines are falling together at Raman shifts below 200 era-1.

longitudinal and transverse modes of one dimensional hydrogen-bonded chains. For the longitudinal modes each "amide" group is assumed to be a point mass located at its center of mass. In the transverse mode, the oxygen and the nitrogen atoms of the amide groups oscillate transverse to the chain of amide groups, and in the longitudinal mode, the point mass amide groups oscillate parallel to the chain of amide groups. Every point mass at the Brillouin zone boundary oscillates 180 ° out of phase with its two nearest neighbours in both modes. According to the conservation of linear momentum, only the zerowavevector modes are expected to appear in Raman spectroscopy. However, this rule is not strictly valid for conglomerates with only a limited number of units

in a chain. Using reliable force constants for the hydrogen bond force constants, the density of states curves for the transverse and the longitudinal modes both show maxima between 100 cm -1 and 200 cm -1, with the maximum for the transverse mode occurring at a slightly higher frequency. Although this solid phase density of states description is only approximate for Raman spectroscopy [32], it is interesting to note that a reasonable qualitative agreement with the experimental results is obtained. The second model used to describe these lowfrequency spectra was a damped oscillator model [30]. It is assumed that the important features of the spectra come from a damped oscillatory character of the angular velocity correlation function in this simple model. The third model used to describe these low-frequency spectra was the Gaussian cage model [33]. In this model, the liquid or solid sample is composed of dipoles having reorientational motion which is greatly restricted by the surrounding molecules, in the high torque limit. The R(~,) representation of the spectra of aprotinin powder and a 20% ( w / w ) aprotinin aqueous solution are shown in Fig. 9A and B, respectively, along with their best-fit damped oscillator and Gaussian cage model curves, both shown in their R(~) representations. Since different scaling factors are used for the various curves, one should not compare absolute intensities in Fig. 9A and B. Table 1 contains the parameters used in the best-fit curves for the damped oscillator and Ganssian cage models, along with the corresponding ( N 2) torque parameter for the Gaussian cage model, for the aprotinin sampies studied. It is somewhat surprising that the torque parameter in the Gaussian cage model appears to in-

Table 1 Best-fit parameters for the R(~) representation of the Gaussian cage model and damped oscillator model fits to the low-frequency aprotinin Raman spectra in the R(~) representation Gaussian cage model a

Damped oscillator model b

f~o (cm- 1)

A(cm- 1)

( N 2)

Ko(0)(cm- 2)

~(cm- 1)

590 580 580

126 140 140

82.5 80.7 80.7

442 000 442 000 442 000

76.4 81.2 81.2

555

155

75.4

500 000

118.3

Aqueous solutions 20% Aprotinin 10% Aprotinin 5% Aprotinin

Powder Aprotinin a Ref. [33]. b Ref. [30].

S.E. May Colaianni et al. / Vibrational Spectroscopy 9 (1995) 111-120

crease from powder to solution, while the damping term in the damped oscillator model decreases. These results are certainly consistent, but the opposite would have been expected. The real Raman spectrum, including the Rayleigh line, takes into account intermolecular dynamics on all time scales. A simulation of this must include a central Lorentzian Rayleigh line. However, fits of a Lorentzian line plus either a damped oscillator or a Gaussian cage model curve gave unsuccessful fits to the real Raman spectrum. This confirms that the R(~) representation is of particular importance in studies of the fast intermolecular dynamics [32]. Inelastic neutron scattering (INS) has been used to study the low-frequency vibrational density of states curves of BPTI, in both the solid state and in aqueous solutions [10]. These spectra also show broad features, with a maximum at 100 cm-1 and another sharper band at approximately 300 cm -1. The band at about 100 cm-1 is similar to the band observed in the R(~) representation of the Raman spectrum. Differences at higher frequencies between the inelastic neutron data and the Raman spectra are not surprising, taking into account the different mechanisms of the two experimental techniques. Thus, the rather sharp band observed around 300 cm -1 could easily be assigned to CH 3 torsions, which give intense bands in the INS spectrum, while these modes are inactive in the Raman spectrum. The INS data were treated theoretically in different harmonic normal mode calculations [10]. Both the low-frequency Raman data and the INS data of other proteins show a broad contourless low-frequency band, which seems to be fairly independent of the protein studied [25,34]. Raman spectroscopy [13,39-43] and computational methods [11,44] have been used to study the low-frequency region of nucleic acids. FIR spectroscopy has been used in low-frequency studies [12,38]. Of particular interest is the application of synchrotron radiation as an excitation source in FIR spectroscopy. A few investigations have already been performed [35-37].

119

with /3-sheet, a-helical and random coil conformations. In H20 and D20 solutions, the Raman spectra show slightly less fl-sheet structure, and more random coil structure, although the /S-sheet structure is still predominant. Only rather concentrated aqueous solutions (2-20%) were investigated by the conventional Raman technique. A windowless cell greatly improved the quality of the NIR-FT-Raman spectra of both the lysozyme and aprotinin solutions. The lysozyme solution showed an intense fluorescence using visible excitation, which disappeared when using NIR excitation, as expected. The synthetic form of BPTI, aprotinin, did not show broad background features with visible excitation, as previously reported for the natural form Trasylol. The conformation of the sulphur bridges is investigated, as is the hydrogen bonding of tyrosine. The overall conclusion is that no significant conformational changes occur in going from solid to solution, although the content of random coil seems to increase somewhat in the aqueous solution, as compared to the solid state. One problem with using NIR excitation is that heating of coloured solid samples can occur, giving a strong background due to the thermal emission. A wavelength around 800 nm should be optimal for avoiding heating. SER spectra on colloidal silver show that it is possible to obtain spectra of PNA in a very diluted aqueous solution. This opens up the possibility of investigating the interaction between DNA and PNA using the SERS technique. The low-frequency part of the vibrational spectrum of aprotinin has been studied, both as a powder and in aqueous solution. With a careful correction for instrumental response, the low-frequency limit in the NIR-FT-Raman spectrum is around 80 cm-1. Different theoretical models are used to account for a rather broad band with a maximum at approximately 100 c m - 1.

Acknowledgements 4. Conclusion In accordance with x-ray studies of solid aprotinin [7,20], the Raman spectra show a secondary structure

The authors are grateful to Haldor Topsoe A / S and to the Danish Natural Science Research Council for funding the NIR-FT-Raman instrument within the Material Technology Development Program. A gen-

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S.E. May Colaianni et al. / Vibrational Spectroscopy 9 (1995) 111-120

eral funding from the Danish Natural Science Research Council supported the project and made the stay for SEMC at Chemical Laboratory V possible. We wish to thank Professor Ole Buchardt and Dr. P.E. Nielsen, University of Copenhagen, for giving us the PNA sample as a gift. Dr. Daniel H. Christensen is thanked for stimulating discussions and for assistance with obtaining the NIR-FT-Raman spectra. Mrs. Ingelise Blangsted is thanked for obtaining the Vis-Raman spectra. OFN thanks the French Ministry of Foreign Affairs for travel support.

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