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[22] Infrared and laser raman spectroscopy
[22]
INFRARED AND LASER RAMAN SPECTROSCOPY
[ 22 ] Infrared
and Laser Raman
247
Spectroscopy
By DONALD F. HOELZL WALLACH and ALLAN ROY OSEROFF
The principles and general applications of infrared spectroscopy have been twice covered in this series 1,2 and are well treated in several specialized books. 3," This chapter will therefore restrict itself to the application of infrared spectroscopy in the dynamic structural analysis of biomembranes, at the same time introducing a related technique, laser-Raman spectroscopy, 5s which offers great future potential. Chemical bonds stretch, twist, rotate, and otherwise vibrate at frequencies of 101° to 1011 Hz. This changes their dipole moments, permitting absorption of electromagnetic radiation of similar, i.e., infrared frequencies. However, the infrared bands observed experimentally often do not represent a single type of bond oscillation, but also reflect coupling between neighboring bonds. Thus the infrared spectrum of a polypeptide is characterized by a set of absorption regions, each representing the combined but variable contributions of the peptide C = O , C - - N , and N - - H , and some also reflecting the folding and H-bonding of the peptide chain. R a m a n scattering also arises from molecular rotations and vibrations. When transparent substances are irradiated with monochromatic light, most of this is transmitted, some is scattered at the incident frequency as Rayleigh scattering, and traces are scattered at higher or lower frequencies as R a m a n scattering. The lrequency shiIt between the incident light and a R a m a n line corresponds to the vibrational frequency of the Raman-active bond. R a m a n scattering arises from the vibrational modulation of bond polarizability, and the intensity of a R a m a n line is roughly proportional to the electron density of the valence bond. ~,s For example, 1D. L. Wood, this series, Vol. 4, p. 104. -~H. Susi, this series, Vol. 26, p. 455. S"Applied Infrared Spectroscopy" (D. N. Kendall, ed.). Van Nostrand-Reinhold, Princeton, New Jersey, 1966. 4H. Susi, in "Structure and Stability of Macromolecules" (S. N. Timasheff and G. D. F. Asman, eds.), p. 575. Dekker, New York, 1969. 5L. A. Woodward, in "Raman Spectroscopy Theory and Practice" (H. A. Szymanski, ed.), p. I. Plenum, New York, 1967. 6D. F. Wallach, J. M. Graham, and A. Oseroff, FEBS (Fed. Eur. Biochem. Soc.) Lett. 7, 330 (1970). 7 M. C. Tobin, "Laser Raman Spectroscopy." Wiley (Interscience), New York, 1971. 8G. J. Thomas, Jr., in "Physical Techniques in Biological Research" (G. Oster, ed.), 2nd ed. Vol. 1A, Chapter 4. Academic Press, New York, 1971.
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the scattering due to S--S stretching is stronger than that due to O---O, and the intensity of the C z C stretch is about twice that of the C - - C mode. s Bending vibrations scatter weakly and, although the O - - H and O--ZH bending modes cause intense infrared absorption, their small Raman cross section allows spectroscopy of dilute aqueous solutions in frequency regions inaccessible to infrared measurements2 Raman spectroscopy is not a new technique. Indeed, Edsall 9 very early employed Raman analyses to characterize the ionic properties of amino acid and dipeptides, using a mercury arc as light source. However, the more extensive use of the method and its application to macromolecules, as well as macromolecular aggregates became feasible only after stable lasers emerged as practical, monochromatic light sources of high intensity. Infrared and Raman spectroscopy allow one to identify, characterize, and quantify specific chemical groupings and to elucidate their interactions with other nearby bonds. Infrared spectroscopy suffers the disadvantage that water dissolves many cell windows and is, moreover, opaque over much of the infrared, due to the intense absorption of the O - - H stretching vibration. This can be avoided by substituting 2HzO for H20, the greater mass of 2H lowering the ~H--O stretching frequency well below the H - - O stretching band of H~O. ~ However, 2H~O can act as a perturbant of macromolecular structure, both through its influence on hydrogen bonding and its entropic effects. Water interference is much less of a problem in Raman spectroscopy and, in infrared analyses of membranes, has been side-stepped by the use of dried films and the recently improved technique of frustrated multiple internal reflection. The latter allows the spectroscopic analysis of samples too opaque for transmission measurements. Reflecting plates giving 25 internal reflections are used in conjunction with a reference beam attenuator. The cost of such an attachment is in the order of $1500. A relative disadvantage of infrared and Raman spectroscopy is the large cost of the high quality instruments desirable for membrane work. Thus, infrared spectroscopy would require an investment of $25,000 for the basic instrument and accessories. Raman spectrophotometers, including the necessary accessories currently cost nearly $45,000, but, because of intense industrial competition and progress, these instruments will shortly fall into the same price category as good infrared spectrophotometers. 9"Amino Acids and Peptides" (E. J. Cohn and J. T. Edsall, eds.), in Proteins, Amino, Chapter 2. Van Nostrand-Reinhold, Princeton, New Jersey, 1943.
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Infrared Spectroscopy The table lists most important group vibrations for membrane biology. Many instruments operate satisfactorily in the 2 0 0 0 - 1 0 0 0 cm -1 range, but only few reach the far infrared, which contains much information concerning the conformations of membrane peptides and the configurations of membrane lipids. In all instances, a resolution of better than 2 cm -1 is desirable and frequency precision must be checked regularly with standard polystyrene films. Scanning speeds should not exceed 4 cm -~ per second. Base lines should be checked routinely, using either matched AgC1 disks or the infrared liquid absorption cells containing 2H20 (to minimize reflections from the 2 windows) in both sample and reference beams. The maximum drift of the base lines should not exceed 0.02 absorbance unit in the region of interest. We do not have experience with all instruments available commercially, but obtain adequate sensitivity and resolution only with instruments in the category of the Perkin Elmer 521 or 621 spectrophotometers. MAJOR INFRAREDABSORPTIONBANDSOF BIOMEMBRANESa Frequency (cm-1) 600 620 650 700 700 720 970 980-1050 1250 1250-1350 1550 1600 1630 1640 1652 1656 1685 1720-1740 2560-2700 2855 2930
Name
Comment
Amide VI Amide V Amide V Amide V Amide IV
C~O out of plane bending N--H out of plane bending, f~-conformation Same, unordered Same, a-helix O ~ C - - N bend CH2-rocking with at least carbons all-trans Phosphatide, unassigned P--O--C stretch C--N stretch (35%); N--H in plane bending (29%) P----O stretching; H-bonding-sensitive N--H in plane bend (63%) and C--N stretch (44%), COOC--O stretch; fl structure Sphingomyelin amide C~O stretch; a-helix C--O stretch; unordered Antiparallel COOH; ester C~O P--O--H stretch Symmetric methylene stretch Asymmetric methylene stretch
Amide III Amide II Amide I Amide I Amide I
a All frequencies were obtained from solid films. AgC1 can be used as support to 400 cm-1.
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Sample Preparation [or Film Infrared Spectroscopy
Essentially all infrared work on membranes has been with dried films or ZH20 suspensions. For the former, large membrane fragments, such as erythrocyte ghosts TM or mitochondrial membranes, 11 freed of nonmembranous components, are pelleted at 2000 g,L,- (15 minutes, 4°; e.g., Sorvall centrifuge RC 2B), washed twice in appropriate, cold buffer (e.g., 7 mM phosphate, pH 7.4) and finally suspended in this medium at a concentration of about 2 mg/ml. Smaller membrane fragments, such as plasma membrane or endoplasmic reticulum, 12,~:~ are treated similarly, but centrifuging is at 100,000 g~,. (40 minutes, 4°; e.g., Spinco ultracentrifuge). Measurements on Lyophilized Films
After centrifugal washing and concentration, the membrane suspensions are applied to AgC1 disks. These are always kept in the dark when not used, or in subdued light otherwise. Slight roughing of new disks with diamond cloth usually improves adherence of aqueous suspensions to the hydrophobic AgCI. Twenty-five ~1 of the membrane suspension is deposited atop the disk, in a layer 20 mm x 8 mm, 1 mm, centered on the disk. A precisely dimensioned template is used to ensure reprodacibility of the films. The layered suspension is frozen rapidly without disturbance of the dimensions of the film, by plunging the disk in liquid nitrogen ( - 1 8 0 ° ) : then the film is lyophilized and stored in vacuo, and in the dark until analyzed. In studies of the effects of various additives, 5 t~l of the agent in a suitable medium (phosphate buffer for ATP, Mg ~÷, Na ÷, K +, EDTA, or combinations thereof, H~O for H ÷ or OH-) are added to the membrane suspensions on the disk; mixed in rapidly and well, and the whole is maintained at the desired temperature for 2 minutes or more prior to freezing. The dried films are placed into the spectrophotometer with their long axes parallel to the beam, and matched AgCI disks, of identical cross section, supporting dried films of identical solution composition are used in the reference beam. Aligned, matched standard films stacked in series (up to 4) give a precise linear relationship between absorbance and peptide concentration in the beam at 1652 cm -1, for s-helical a n d / o r unordered peptides. 1° After lyophilization, the films are very fragile and need delicate handling to avoid disruption. 10j. M. l~J. M. riD. F. 13D. F.
Graham and D. F. H. Wallach, Biochim. Biophys. Acta 193, 225 (1969). Graham and D. F. H. Wallach, Biochirn. Biophys. ,4cta 241, 380 (1971). H. Wallach and P. H. Zahler, Proc. Nat. Acad. Sci. U.S. 56, 1552 (1966). H. Wallach and P. H. Zahler, Biochim. Biophys. Acta 105, 186 (1968).
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Measurements in 2H20 The O - - H deformation band absorbs very strongly between 1700 and 1600 cm -1. However, this region is transparent in D20, whose O - - D deformation band lies near 1200 cm -1. Except for these regions both H20 and DzO exhibit about 40% transmittance and can be used in highquality double-beam spectrometers. For measurements in D20, large and small membrane fragments are sedimented from their respective media as before, the supernatant is removed meticulously, and the pellets are dispersed in appropriate buffers (e.g., 7 m M phosphate, in 2H20, pH 7.4) in a long centrifuge tube, using a 1-ml disposable syringe with a 25-gauge needle. Foaming is avoided by expelling the syringe contents tangentially to the wall of the tube. After 15 minutes at 1°-0 ° in closed tubes, the membranes are repelleted at appropriate speeds and the '-'HzO wash is repeated twice. The particles are finally dispersed thoroughly in an appropriate ~H20 medium to a concentration of about 3 mg of protein per milliliter. About 0.2 ml of the membrane suspension is taken up into a 1-ml disposable syringe and introduced via a Luer injection port into a dry infrared liquid absorption cell, path length 0.5 mm, equipped with I R T R A N 2 windows; bubbles must be avoided. An identical matched cell containing an appropriate 2H20 solution is used as a reference. Cells with I R T R A N windows must be matched to 1% of path length or better to avoid interference fringes due to light reflected from the various window surfaces. This problem is less severe with CaF windows. All additions made to both sample and reference cells are to the same final concentrations. Analysis of Results TM Soluble Polypeptides and Proteins. Several infrared bands (see table) reflect conformation. Thus, poly-e-lysine can exist in one or a mixture of conformations--unordered at pH 7.0 and low ionic strength, a-helical at pH 11.3 at 20 °, and /3-structured (antiparallel) at pH 11.5, 55°. 1~ Infrared spectroscopy distinguishes the latter two, because the Amide I band lies at 1652 cm -I in a-helices and near 1625 cm -1 in fl-structures. The antiparallel fl-conformation also causes appearance of a shoulder at 1693 cm -1. However, in the spectral region of 1700-1600 cm -1, one cannot discriminate between a-helix and unordered conformations, except when the involved peptides can be placed in a fixed, known orientation ,4 In infrared and Raman spectroscopy the synthetic standards usually employed cannot be taken as absolute or ideal reference materials any more than in optical activity measurements, and the experimenter must exercise appropriate caution in the interpretation of data from biological materials. 'ST. Miyazawa and E. Blout, J. Amer. Chem. Soc. 83, 712 (1961).
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CHARACTERIZATION OF MEMBRANES AND COMPONENTS
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relative to the polarization plane of a polarized infrared beam (infrared dichroism); this is rarely feasible. This lack of discrimination is due in part to side chain absorption, which obscures the splitting expected from the fact that the Amide I band of pure unordered peptides lies at 1656 em -1, and at 1652 cm -1 for pure a-helix. However, the conjoint presence of fl-structures plus a-helical a n d / o r unordered conformations can be recognized. Thus, lysozyme, ( 1 0 % antiparallel-fl), TM shows a 1650 cm -~ peak with 1632 cm -~ shoulder, while carboxypeptidase A with (15% fl) has a 1650 peak and a 1635 cm -~ shoulder. 17 The Amide V band also mirrors 2 ° structure in that extendedunordered and a-helical poly-~,-methyl-L-glutamate, absorb at 700, 650, and 620 c m -1, respectively. TM Concordantly, lysozyme, a mixture of fl-structured, unordered, and helical peptide has three bands in this region, at 690, 650, and 600 cm -~. Membrane Proteins. Infrared spectra of resting erythrocyte ~ or other animal plasma membranes, 12,1~ dried from neutral aqueous solutions or suspended in neutral 2H20 show the Amide I bands at 1652 cm -1, due to a-helical a n d / o r unordered conformations, but microbial outer membranes also seem to contain some fl-structure. ~9,2° Normally, inner mitochondriaI membranes contain significant antiparallel fl-structured peptide. 21 Their film or 2H20 spectra peak near 1650 cm -1, but show a pronounced shoulder near 1635-1640 cm -~ and a smaller one near 1690 cm -~. The presence in membranes of ordered conformations other than a-helix is not surprising, since most proteins are conformational mixtures, where even the ordered segments deviate from ideal synthetic models.
Inlrared Detection o] Con]ormation Change in Membrane Proteins. For rapid metabolic measurements, buffered membrane suspensions, on AgC1 disks, are quickly frozen in liquid N~, lyophilized, and their infrared spectra measured. Some sensitive membrane functions can be recovered ~0,1~ provided the lyophilized membranes are first rehydrated at 100% relative humidity for 1-2 hours at 1 °-4° prior to suspension in aqueous buffer. Erythrocyte ghosts, thus treated, reform biconcave disks, with full Na÷-K+ATPase activity and similar treatment of lyophilized mitochondria yields almost full recovery of coupled oxidative phosphorylation, zl Lipids. The table lists some of the vibrations obtained from phospho'eD. C. Phillips, Sci. Amer. 215, 78 (1966). l ' W . N. Lipscomb, Accounts Chem. Res. 3, 81 (1970). 1ST. Miyazawa, Y. Masua, and K. Fukushima, 1. Polymer Sci. 62, 562 (1962). a°D. H. Green and M. R. J. Salton, Biochim. Biophys. Acta 9,11, 139 (1970). 2oG. L. Choules and R. F. Bjorklund, Biochemistry 9, 4759 (1970). " D. F. H. Wallach, J. M. Graham, and B. R. Fernbach, Arch. Biochim. Biophys. 131, 322 (1969).
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lipids. CH~ rocking (720 cm -1) indicates interaction between adjacent apolar chains, when at least 4 carbon atoms lie in an all-trans-planar configuration; in solids this band splits by as much as 6 cm -1. P - - O stretching (1250-1250 cm -1) can decrease in frequency and intensify upon H-bonding. The major lipid contribution near the Amide I region is due to sphingosine, whose amide absorbs at 1640-1540 cm -1, and phosphatidylserine - - C O 0 - , absorbing near 1600 cm -~. In addition all carboxylic esters absorb very strongly near 1730 c m -1, and this band can be used to monitor phosphatide extraction from membranes. Aqueous dispersions of myelin, as well as membrane lipid extracts exhibit prominent CH~ rocking at 720 cm -x, but this is lacking in plasma membranes at room temperature, 2~ possibly because of apolar lipid protein associations. In Micrococcus lysodeikticus, CH2 rocking, as well as the ratio of the intensities of the asymmetric/symmetric methylene stretching modes vary in whole membranes, their lipid extracts and the extracted residues (0.3, 0.1, and 1.4, respectively, at - 1 8 0 ° ) . Only extracted lipid shows significant CHz-rocking and the data all suggest apolar lipid-protein interactions. ~9 P : O stretching in dried red cell ghosts lies at 1225 cm -~, as does that of D20 dispersions of the membrane lipids. If these groups were ionically linked to membrane proteins, the P - - O - phosphorus would be more electropositive, changing P : O stretching accordingly; however, this is not seen. The lower P : O stretching value than found in pure, isolated phospholipids suggests H-bonding. Pertinently, studies on the P : O stretching and CH2 rocking amplitudes in cephalin and lecithin films~3 show intensification of these bands with applied electric fields, due to alignment of the phosphate-base dipoles and secondary rearrangement of the hydrocarbon chains; this shows that fatty acids may reorient with changing transmembrane potentials. R a m a n Spectroscopy General Observations The basic Raman spectrometer suitable for biological work consists of (1) a stable gas laser source, (2) a double monochromator, (3) an optical system to focus the laser beam on or into the sample, as well as to collect and image the scattered radiation on the monochromator slit, and (4) a cooled, low noise photomultiplier capable of resolving and counting single photons. Unless computer facilities are available, a multi22D. Chapman, V. B. Kamat, and R. J. Levene, Science 160, 314 (1968). 2~L. May, A. B. Kamble, and I. P. Acosta, J. Memb. Biol. 2, 192 (1970).
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channel analyzer, or signal averager is a desirable option for high sensitivity measurements, and a necessity for differential spectroscopy. The instrument can be purchased as a complete package from a number of commercial sources, or, for additional flexibility or special purposes, may be assembled from the individual components. In specifying the instrument, an important decision is the choice of laser source. With samples absorbing near 5000 A, a krypton ion laser with lines at 5682 and 6471 A is useful. More generally, the best choice is an argon ion laser operated at 5145 or 4 8 8 0 A with as high an output power as can be tolerated by the sample and by the budget of the experimenter; at these wavelengths the efficiency of the detector is great, as is the Raman scattering cross section. '~ High power levels are necessary for work with dilute solutions. In the absence of strong fluorescence, the signal-to-noise ratio of a Raman band will be approximately proportional to the square root of the product of laser power, sample concentration, and measurement time. As a rough guide, reasonable spectra of 10 mg/mt polypeptide or protein solutions require about 1 W of 5145 A laser power at a scan rate of 25 cm-' per minute. Fluorescence is a common complication in laser Raman spectroscopy and requires careful sample and reagent purification. Nevertheless, a significant fluorescence background often remains, which may initially be of comparable intensity using either 5145 or 6471 A excitation. However, after prolonged exposure to the beam, the fluorescence gradually decreases. 2'-'-'7 The degree and rate of the fluorescence inactivation are maximal at the shorter laser wavelengths (e.g., 4880 and 5145 A) and at high laser power; these conditions should be used when possible. The presence of fluorescence indicates absorption of light, some of which is dissipated as heat and can cause localized heating and sample damage. In control studies of erythrocyte membrane suspensions in our laboratory, using 1.5 W of 4880 A laser power we found a rapid decline in Na+-K+-ATPase activity, paralleled by a decrease in fluorescence. The enzyme damage was potentiated by oxygen; in deoxygenated samples the ATPase activity remained constant and the initial fluorescence was significantly reduced.
Sample Preparation Because of fluorescence and Tyndall scattering from inhomogeneities and particulate impurities, sample quality is more critical in Raman than in infrared spectroscopy. The general precautions discussed in chapters 2*R. 2~R. ~B. 27A.
C. Lord and N. T. Yu, J. Mol. Biol. 50, 509 (1970). C. Lord and N. T. Yu, 1. Mol. Biol. 51, 203 (1970). L. Tomlinson and W. L. Peticolas, J. Chem. Phys. 52, 2154 (1970). Lewis and H. A. Scheraga, Macromolecules 4, 539 (1971).
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on light scattering 2s and fluorescence -°9 in this series should be followed. All chemicals should be of highest available quality. Distilled, degassed water is used, and if solvent fluorescence is apparent further purification may be necessary. ~ Samples may be purified by centrifugation a n d / o r filtration through activated charcoal. Where possible, samples should be filtered through a 0.05-/~m Millipore filter while filling the cell, using 1-ml disposable syringes. Gas bubbles and dust on the inside walls of the cell should be avoided. For measurements in -~H~O, the usual precautions must be followed to avoid contamination by H20. Cacodylate is a useful buffer because it has a very weak Raman spectrum with the exception of a line near 600 cm-1. 26,:" Measurements of Solutions or Suspensions Vertical illumination should be used for clear to moderately turbid solutions or suspensions, with the laser beam focused through a bottom window into the cell, and the scattered radiation collected from the side at 90 ° to the beam. Cells should be made of nonfluorescent fused quartz, with a square or cylindrical cross section, and a flat, well polished bottom. Typical dimensions are 2 mm wide and 15 mm high, but these are not critical. The top of the cell is sealed with a transparent cap to permit deoxygenation and to keep out dust. The cell should be completely filled to avoid background reflections from the curved air-liquid interface. For temperature measurements and for long-term sample stability, the cell should be jacketed or mounted in a massive, temperature-controlled holder. :~° If a jacket is used, the circulating liquid should not pass over the entrance or collection windows. In normal operation, the monochromator collects only the radiation scattered from the cylindrical volume illuminated by the focused laser beam; this is about 2 0 - 5 0 t~m in diameter and 2 - 1 0 mm high. Turbid samples diffuse the beam, as well as the Raman radiation, into a much larger volume so that the number of signal photons which reach the monochromator is greatly reduced. At the same time, noise is increased because of the extensive Tyndall scattering. In such cases we find the following procedure to be effective: The cell is raised so that its bottom is at the midline of the collecting lens and positioned so the laser beam barely grazes the inner surface of the wall closest to the lens. The focusing lens is adjusted so that at the bottom of the cell the beam appears as a bright, sharply defined cylinder, 2-3 mm high and as narrow as possible. The height of the monochromator slit is decreased until the bright image ~"M. Bier, this series, Vol. 4, p. 147. ~ D. J. R. Laurence, this series, Vol. 4, p. 174. ~E. W. Small and W. L. Peticolas, Biopolymers 10, 1377 (1971).
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CHARACTERIZATIONOF MEMBRANES AND COMPONENTS
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is centered on, but slightly overlaps, the top and bottom of the slit. With care, we have obtained good spectra from suspensions of erythrocyte ghosts (up to 5 m g / m l protein) and B-structured poly-L-lysine (up to 10 m g / m l ) . 6
Differential Spectroscopy In direct measurements of dilute solutions, the broad water background scattering at 1640 cm -1 begins to obscure the solute spectrum at sample concentrations less than 10-20 mg/ml. We have found that the solvent background can be removed by means of differential spectroscopy and that good spectra can be resolved in the Amide I region at concentrations below 1 mg/ml. 6 Our prototype spectrometer uses an oscillating, partitioned cell containing sample and reference solutions in tandem. As the sample (reference) compartments pass alternately through the fixed laser beam, the number of detected photons is added (subtracted from) the memory of a multichannel analyzer, which is synchronized with the cell motion. The spectrometer is null balanced on the strong 3500 cm -1 water band or on an internal reference line in two steps: First, the relative time spent by the laser beam in each compartment during a cycle is mechanically adjusted by shifting the midpoint of the oscillation with respect to the fixed beam. Final balance is through electronic adjustment of the add or subtract periods during which counts are accepted by the analyzer (usually 1 second each). An auxiliary, fixed-wavelength spectrometer monitors the Raman scattering during the course of a measurement and maintains the electronic null, thus compensating for changes in turbidity. This technique allows resolution of defined spectra from structured backgrounds twenty times as large, and is, therefore, capable of separating overlapping bands in complex, multicomponent systems.
interpretation 31 Proteins and Polypeptides. In studying polypeptide solutions, Raman spectroscopy is superior to infrared absorption measurements because it can discriminate between a-helical, B, and random coil conformations. 6,27,~'-'-3' For example, in the Amide I region the three types of struc~1In infrared and Raman spectroscopy the synthetic standards usually employed cannot be taken as absolute or ideal reference materials any more than in optical activity measurements and the experimenter must exercise appropriate caution in the interpretation of data from biological materials. a2M. Smith, A. G. Walton, and J. L. Koenig, Biopolymers 8, 29 (1969). nJ. L. Koenig and P. L. Sutton, Biopolymers 8, 167 (1969); 9, 1229 (1970); 10, 89 (1971). 84p. Sutton and J. L. Koenig, Biopolymers 9, 615 (1970).
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ture can be identified in poly-L-lysine solutions by bands at 1647, 1672, and 1665 cm -1, respectively. 6 Quantitative determination of complex protein structure is difficult, 3~ but it is already apparent that R a m a n spectroscopy can provide at least as much information as can be extracted from optical activity measurements, 27 and is more amenable to theoretical analysis, as well as to kinetic and metabolic applications. While Amide I I scattering is very weak, z*,2~,32-34 the Amide I I I spectrum is readily visible and is more complex than in the infrared. In polypeptides it has been assigned to bands near 1320 cm-1, 2m3-35 and in proteins to a conformation-sensitive region near 1250 cm-1. 24,2~,3~,36 Because of the strength of the S - - S scattering, the disulfide modes are easily observed. 2',3~ The number of bonds is given by the intensity of a line near 509 cm -~, while the ratio of this peak to the C - - S - - S line at 661 cm -1 has been related to the C - - S - - S - - C dihedral angle. 2~,3~
Lipids In aqueous multilayer suspensions of dipamitoyllecithin, the intensity of bands near 1066 and 1130 cm -1 has been taken to a measure of the amount of all-trans structure in the paraffin chains, while a broad band at 1089 cm -1 is assigned to the appearance of structures containing several gauche rotations of the melted paraffin2 ~ The ratios of the 1089:1128 cm -1 or 1089:1066 cm -1 bands provide a sensitive probe of the extent of liquid crystallike order and the cooperativity of the melting transition. F o r the more complex system of the erythrocyte membrane our preliminary studies have shown that P - - O - - C and C z O lipid modes can be resolved together with peptide bands from the protein moiety. Acknowledgments Supported by Grant No. 84750-0 from the U.S. Public Health Service, the Tufts-New England Medical Center Department of Therapeutic Radiology Grant from the National Cancer Institute, and a AFOSR-NRC Fellowship.
~R. C. Lord, Proc. Int. Congr. Pure Appl. Chem. 33(7), 179 (1971). A. M. Bellocq, R. C. Lord, and R. Mendelsohn, Biochim. Biophys. Acta in press. 3, j. L. Lippert and W. L. Peticolas, Proc. Nat. Acad. Sci. U.S. 68, 1572 (1971).
INFRARED AND LASER RAMAN SPECTROSCOPY
[ 22 ] Infrared
and Laser Raman
247
Spectroscopy
By DONALD F. HOELZL WALLACH and ALLAN ROY OSEROFF
The principles and general applications of infrared spectroscopy have been twice covered in this series 1,2 and are well treated in several specialized books. 3," This chapter will therefore restrict itself to the application of infrared spectroscopy in the dynamic structural analysis of biomembranes, at the same time introducing a related technique, laser-Raman spectroscopy, 5s which offers great future potential. Chemical bonds stretch, twist, rotate, and otherwise vibrate at frequencies of 101° to 1011 Hz. This changes their dipole moments, permitting absorption of electromagnetic radiation of similar, i.e., infrared frequencies. However, the infrared bands observed experimentally often do not represent a single type of bond oscillation, but also reflect coupling between neighboring bonds. Thus the infrared spectrum of a polypeptide is characterized by a set of absorption regions, each representing the combined but variable contributions of the peptide C = O , C - - N , and N - - H , and some also reflecting the folding and H-bonding of the peptide chain. R a m a n scattering also arises from molecular rotations and vibrations. When transparent substances are irradiated with monochromatic light, most of this is transmitted, some is scattered at the incident frequency as Rayleigh scattering, and traces are scattered at higher or lower frequencies as R a m a n scattering. The lrequency shiIt between the incident light and a R a m a n line corresponds to the vibrational frequency of the Raman-active bond. R a m a n scattering arises from the vibrational modulation of bond polarizability, and the intensity of a R a m a n line is roughly proportional to the electron density of the valence bond. ~,s For example, 1D. L. Wood, this series, Vol. 4, p. 104. -~H. Susi, this series, Vol. 26, p. 455. S"Applied Infrared Spectroscopy" (D. N. Kendall, ed.). Van Nostrand-Reinhold, Princeton, New Jersey, 1966. 4H. Susi, in "Structure and Stability of Macromolecules" (S. N. Timasheff and G. D. F. Asman, eds.), p. 575. Dekker, New York, 1969. 5L. A. Woodward, in "Raman Spectroscopy Theory and Practice" (H. A. Szymanski, ed.), p. I. Plenum, New York, 1967. 6D. F. Wallach, J. M. Graham, and A. Oseroff, FEBS (Fed. Eur. Biochem. Soc.) Lett. 7, 330 (1970). 7 M. C. Tobin, "Laser Raman Spectroscopy." Wiley (Interscience), New York, 1971. 8G. J. Thomas, Jr., in "Physical Techniques in Biological Research" (G. Oster, ed.), 2nd ed. Vol. 1A, Chapter 4. Academic Press, New York, 1971.
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the scattering due to S--S stretching is stronger than that due to O---O, and the intensity of the C z C stretch is about twice that of the C - - C mode. s Bending vibrations scatter weakly and, although the O - - H and O--ZH bending modes cause intense infrared absorption, their small Raman cross section allows spectroscopy of dilute aqueous solutions in frequency regions inaccessible to infrared measurements2 Raman spectroscopy is not a new technique. Indeed, Edsall 9 very early employed Raman analyses to characterize the ionic properties of amino acid and dipeptides, using a mercury arc as light source. However, the more extensive use of the method and its application to macromolecules, as well as macromolecular aggregates became feasible only after stable lasers emerged as practical, monochromatic light sources of high intensity. Infrared and Raman spectroscopy allow one to identify, characterize, and quantify specific chemical groupings and to elucidate their interactions with other nearby bonds. Infrared spectroscopy suffers the disadvantage that water dissolves many cell windows and is, moreover, opaque over much of the infrared, due to the intense absorption of the O - - H stretching vibration. This can be avoided by substituting 2HzO for H20, the greater mass of 2H lowering the ~H--O stretching frequency well below the H - - O stretching band of H~O. ~ However, 2H~O can act as a perturbant of macromolecular structure, both through its influence on hydrogen bonding and its entropic effects. Water interference is much less of a problem in Raman spectroscopy and, in infrared analyses of membranes, has been side-stepped by the use of dried films and the recently improved technique of frustrated multiple internal reflection. The latter allows the spectroscopic analysis of samples too opaque for transmission measurements. Reflecting plates giving 25 internal reflections are used in conjunction with a reference beam attenuator. The cost of such an attachment is in the order of $1500. A relative disadvantage of infrared and Raman spectroscopy is the large cost of the high quality instruments desirable for membrane work. Thus, infrared spectroscopy would require an investment of $25,000 for the basic instrument and accessories. Raman spectrophotometers, including the necessary accessories currently cost nearly $45,000, but, because of intense industrial competition and progress, these instruments will shortly fall into the same price category as good infrared spectrophotometers. 9"Amino Acids and Peptides" (E. J. Cohn and J. T. Edsall, eds.), in Proteins, Amino, Chapter 2. Van Nostrand-Reinhold, Princeton, New Jersey, 1943.
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Infrared Spectroscopy The table lists most important group vibrations for membrane biology. Many instruments operate satisfactorily in the 2 0 0 0 - 1 0 0 0 cm -1 range, but only few reach the far infrared, which contains much information concerning the conformations of membrane peptides and the configurations of membrane lipids. In all instances, a resolution of better than 2 cm -1 is desirable and frequency precision must be checked regularly with standard polystyrene films. Scanning speeds should not exceed 4 cm -~ per second. Base lines should be checked routinely, using either matched AgC1 disks or the infrared liquid absorption cells containing 2H20 (to minimize reflections from the 2 windows) in both sample and reference beams. The maximum drift of the base lines should not exceed 0.02 absorbance unit in the region of interest. We do not have experience with all instruments available commercially, but obtain adequate sensitivity and resolution only with instruments in the category of the Perkin Elmer 521 or 621 spectrophotometers. MAJOR INFRAREDABSORPTIONBANDSOF BIOMEMBRANESa Frequency (cm-1) 600 620 650 700 700 720 970 980-1050 1250 1250-1350 1550 1600 1630 1640 1652 1656 1685 1720-1740 2560-2700 2855 2930
Name
Comment
Amide VI Amide V Amide V Amide V Amide IV
C~O out of plane bending N--H out of plane bending, f~-conformation Same, unordered Same, a-helix O ~ C - - N bend CH2-rocking with at least carbons all-trans Phosphatide, unassigned P--O--C stretch C--N stretch (35%); N--H in plane bending (29%) P----O stretching; H-bonding-sensitive N--H in plane bend (63%) and C--N stretch (44%), COOC--O stretch; fl structure Sphingomyelin amide C~O stretch; a-helix C--O stretch; unordered Antiparallel COOH; ester C~O P--O--H stretch Symmetric methylene stretch Asymmetric methylene stretch
Amide III Amide II Amide I Amide I Amide I
a All frequencies were obtained from solid films. AgC1 can be used as support to 400 cm-1.
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Sample Preparation [or Film Infrared Spectroscopy
Essentially all infrared work on membranes has been with dried films or ZH20 suspensions. For the former, large membrane fragments, such as erythrocyte ghosts TM or mitochondrial membranes, 11 freed of nonmembranous components, are pelleted at 2000 g,L,- (15 minutes, 4°; e.g., Sorvall centrifuge RC 2B), washed twice in appropriate, cold buffer (e.g., 7 mM phosphate, pH 7.4) and finally suspended in this medium at a concentration of about 2 mg/ml. Smaller membrane fragments, such as plasma membrane or endoplasmic reticulum, 12,~:~ are treated similarly, but centrifuging is at 100,000 g~,. (40 minutes, 4°; e.g., Spinco ultracentrifuge). Measurements on Lyophilized Films
After centrifugal washing and concentration, the membrane suspensions are applied to AgC1 disks. These are always kept in the dark when not used, or in subdued light otherwise. Slight roughing of new disks with diamond cloth usually improves adherence of aqueous suspensions to the hydrophobic AgCI. Twenty-five ~1 of the membrane suspension is deposited atop the disk, in a layer 20 mm x 8 mm, 1 mm, centered on the disk. A precisely dimensioned template is used to ensure reprodacibility of the films. The layered suspension is frozen rapidly without disturbance of the dimensions of the film, by plunging the disk in liquid nitrogen ( - 1 8 0 ° ) : then the film is lyophilized and stored in vacuo, and in the dark until analyzed. In studies of the effects of various additives, 5 t~l of the agent in a suitable medium (phosphate buffer for ATP, Mg ~÷, Na ÷, K +, EDTA, or combinations thereof, H~O for H ÷ or OH-) are added to the membrane suspensions on the disk; mixed in rapidly and well, and the whole is maintained at the desired temperature for 2 minutes or more prior to freezing. The dried films are placed into the spectrophotometer with their long axes parallel to the beam, and matched AgCI disks, of identical cross section, supporting dried films of identical solution composition are used in the reference beam. Aligned, matched standard films stacked in series (up to 4) give a precise linear relationship between absorbance and peptide concentration in the beam at 1652 cm -1, for s-helical a n d / o r unordered peptides. 1° After lyophilization, the films are very fragile and need delicate handling to avoid disruption. 10j. M. l~J. M. riD. F. 13D. F.
Graham and D. F. H. Wallach, Biochim. Biophys. Acta 193, 225 (1969). Graham and D. F. H. Wallach, Biochirn. Biophys. ,4cta 241, 380 (1971). H. Wallach and P. H. Zahler, Proc. Nat. Acad. Sci. U.S. 56, 1552 (1966). H. Wallach and P. H. Zahler, Biochim. Biophys. Acta 105, 186 (1968).
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Measurements in 2H20 The O - - H deformation band absorbs very strongly between 1700 and 1600 cm -1. However, this region is transparent in D20, whose O - - D deformation band lies near 1200 cm -1. Except for these regions both H20 and DzO exhibit about 40% transmittance and can be used in highquality double-beam spectrometers. For measurements in D20, large and small membrane fragments are sedimented from their respective media as before, the supernatant is removed meticulously, and the pellets are dispersed in appropriate buffers (e.g., 7 m M phosphate, in 2H20, pH 7.4) in a long centrifuge tube, using a 1-ml disposable syringe with a 25-gauge needle. Foaming is avoided by expelling the syringe contents tangentially to the wall of the tube. After 15 minutes at 1°-0 ° in closed tubes, the membranes are repelleted at appropriate speeds and the '-'HzO wash is repeated twice. The particles are finally dispersed thoroughly in an appropriate ~H20 medium to a concentration of about 3 mg of protein per milliliter. About 0.2 ml of the membrane suspension is taken up into a 1-ml disposable syringe and introduced via a Luer injection port into a dry infrared liquid absorption cell, path length 0.5 mm, equipped with I R T R A N 2 windows; bubbles must be avoided. An identical matched cell containing an appropriate 2H20 solution is used as a reference. Cells with I R T R A N windows must be matched to 1% of path length or better to avoid interference fringes due to light reflected from the various window surfaces. This problem is less severe with CaF windows. All additions made to both sample and reference cells are to the same final concentrations. Analysis of Results TM Soluble Polypeptides and Proteins. Several infrared bands (see table) reflect conformation. Thus, poly-e-lysine can exist in one or a mixture of conformations--unordered at pH 7.0 and low ionic strength, a-helical at pH 11.3 at 20 °, and /3-structured (antiparallel) at pH 11.5, 55°. 1~ Infrared spectroscopy distinguishes the latter two, because the Amide I band lies at 1652 cm -I in a-helices and near 1625 cm -1 in fl-structures. The antiparallel fl-conformation also causes appearance of a shoulder at 1693 cm -1. However, in the spectral region of 1700-1600 cm -1, one cannot discriminate between a-helix and unordered conformations, except when the involved peptides can be placed in a fixed, known orientation ,4 In infrared and Raman spectroscopy the synthetic standards usually employed cannot be taken as absolute or ideal reference materials any more than in optical activity measurements, and the experimenter must exercise appropriate caution in the interpretation of data from biological materials. 'ST. Miyazawa and E. Blout, J. Amer. Chem. Soc. 83, 712 (1961).
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CHARACTERIZATION OF MEMBRANES AND COMPONENTS
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relative to the polarization plane of a polarized infrared beam (infrared dichroism); this is rarely feasible. This lack of discrimination is due in part to side chain absorption, which obscures the splitting expected from the fact that the Amide I band of pure unordered peptides lies at 1656 em -1, and at 1652 cm -1 for pure a-helix. However, the conjoint presence of fl-structures plus a-helical a n d / o r unordered conformations can be recognized. Thus, lysozyme, ( 1 0 % antiparallel-fl), TM shows a 1650 cm -~ peak with 1632 cm -~ shoulder, while carboxypeptidase A with (15% fl) has a 1650 peak and a 1635 cm -~ shoulder. 17 The Amide V band also mirrors 2 ° structure in that extendedunordered and a-helical poly-~,-methyl-L-glutamate, absorb at 700, 650, and 620 c m -1, respectively. TM Concordantly, lysozyme, a mixture of fl-structured, unordered, and helical peptide has three bands in this region, at 690, 650, and 600 cm -~. Membrane Proteins. Infrared spectra of resting erythrocyte ~ or other animal plasma membranes, 12,1~ dried from neutral aqueous solutions or suspended in neutral 2H20 show the Amide I bands at 1652 cm -1, due to a-helical a n d / o r unordered conformations, but microbial outer membranes also seem to contain some fl-structure. ~9,2° Normally, inner mitochondriaI membranes contain significant antiparallel fl-structured peptide. 21 Their film or 2H20 spectra peak near 1650 cm -1, but show a pronounced shoulder near 1635-1640 cm -~ and a smaller one near 1690 cm -~. The presence in membranes of ordered conformations other than a-helix is not surprising, since most proteins are conformational mixtures, where even the ordered segments deviate from ideal synthetic models.
Inlrared Detection o] Con]ormation Change in Membrane Proteins. For rapid metabolic measurements, buffered membrane suspensions, on AgC1 disks, are quickly frozen in liquid N~, lyophilized, and their infrared spectra measured. Some sensitive membrane functions can be recovered ~0,1~ provided the lyophilized membranes are first rehydrated at 100% relative humidity for 1-2 hours at 1 °-4° prior to suspension in aqueous buffer. Erythrocyte ghosts, thus treated, reform biconcave disks, with full Na÷-K+ATPase activity and similar treatment of lyophilized mitochondria yields almost full recovery of coupled oxidative phosphorylation, zl Lipids. The table lists some of the vibrations obtained from phospho'eD. C. Phillips, Sci. Amer. 215, 78 (1966). l ' W . N. Lipscomb, Accounts Chem. Res. 3, 81 (1970). 1ST. Miyazawa, Y. Masua, and K. Fukushima, 1. Polymer Sci. 62, 562 (1962). a°D. H. Green and M. R. J. Salton, Biochim. Biophys. Acta 9,11, 139 (1970). 2oG. L. Choules and R. F. Bjorklund, Biochemistry 9, 4759 (1970). " D. F. H. Wallach, J. M. Graham, and B. R. Fernbach, Arch. Biochim. Biophys. 131, 322 (1969).
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lipids. CH~ rocking (720 cm -1) indicates interaction between adjacent apolar chains, when at least 4 carbon atoms lie in an all-trans-planar configuration; in solids this band splits by as much as 6 cm -1. P - - O stretching (1250-1250 cm -1) can decrease in frequency and intensify upon H-bonding. The major lipid contribution near the Amide I region is due to sphingosine, whose amide absorbs at 1640-1540 cm -1, and phosphatidylserine - - C O 0 - , absorbing near 1600 cm -~. In addition all carboxylic esters absorb very strongly near 1730 c m -1, and this band can be used to monitor phosphatide extraction from membranes. Aqueous dispersions of myelin, as well as membrane lipid extracts exhibit prominent CH~ rocking at 720 cm -x, but this is lacking in plasma membranes at room temperature, 2~ possibly because of apolar lipid protein associations. In Micrococcus lysodeikticus, CH2 rocking, as well as the ratio of the intensities of the asymmetric/symmetric methylene stretching modes vary in whole membranes, their lipid extracts and the extracted residues (0.3, 0.1, and 1.4, respectively, at - 1 8 0 ° ) . Only extracted lipid shows significant CHz-rocking and the data all suggest apolar lipid-protein interactions. ~9 P : O stretching in dried red cell ghosts lies at 1225 cm -~, as does that of D20 dispersions of the membrane lipids. If these groups were ionically linked to membrane proteins, the P - - O - phosphorus would be more electropositive, changing P : O stretching accordingly; however, this is not seen. The lower P : O stretching value than found in pure, isolated phospholipids suggests H-bonding. Pertinently, studies on the P : O stretching and CH2 rocking amplitudes in cephalin and lecithin films~3 show intensification of these bands with applied electric fields, due to alignment of the phosphate-base dipoles and secondary rearrangement of the hydrocarbon chains; this shows that fatty acids may reorient with changing transmembrane potentials. R a m a n Spectroscopy General Observations The basic Raman spectrometer suitable for biological work consists of (1) a stable gas laser source, (2) a double monochromator, (3) an optical system to focus the laser beam on or into the sample, as well as to collect and image the scattered radiation on the monochromator slit, and (4) a cooled, low noise photomultiplier capable of resolving and counting single photons. Unless computer facilities are available, a multi22D. Chapman, V. B. Kamat, and R. J. Levene, Science 160, 314 (1968). 2~L. May, A. B. Kamble, and I. P. Acosta, J. Memb. Biol. 2, 192 (1970).
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channel analyzer, or signal averager is a desirable option for high sensitivity measurements, and a necessity for differential spectroscopy. The instrument can be purchased as a complete package from a number of commercial sources, or, for additional flexibility or special purposes, may be assembled from the individual components. In specifying the instrument, an important decision is the choice of laser source. With samples absorbing near 5000 A, a krypton ion laser with lines at 5682 and 6471 A is useful. More generally, the best choice is an argon ion laser operated at 5145 or 4 8 8 0 A with as high an output power as can be tolerated by the sample and by the budget of the experimenter; at these wavelengths the efficiency of the detector is great, as is the Raman scattering cross section. '~ High power levels are necessary for work with dilute solutions. In the absence of strong fluorescence, the signal-to-noise ratio of a Raman band will be approximately proportional to the square root of the product of laser power, sample concentration, and measurement time. As a rough guide, reasonable spectra of 10 mg/mt polypeptide or protein solutions require about 1 W of 5145 A laser power at a scan rate of 25 cm-' per minute. Fluorescence is a common complication in laser Raman spectroscopy and requires careful sample and reagent purification. Nevertheless, a significant fluorescence background often remains, which may initially be of comparable intensity using either 5145 or 6471 A excitation. However, after prolonged exposure to the beam, the fluorescence gradually decreases. 2'-'-'7 The degree and rate of the fluorescence inactivation are maximal at the shorter laser wavelengths (e.g., 4880 and 5145 A) and at high laser power; these conditions should be used when possible. The presence of fluorescence indicates absorption of light, some of which is dissipated as heat and can cause localized heating and sample damage. In control studies of erythrocyte membrane suspensions in our laboratory, using 1.5 W of 4880 A laser power we found a rapid decline in Na+-K+-ATPase activity, paralleled by a decrease in fluorescence. The enzyme damage was potentiated by oxygen; in deoxygenated samples the ATPase activity remained constant and the initial fluorescence was significantly reduced.
Sample Preparation Because of fluorescence and Tyndall scattering from inhomogeneities and particulate impurities, sample quality is more critical in Raman than in infrared spectroscopy. The general precautions discussed in chapters 2*R. 2~R. ~B. 27A.
C. Lord and N. T. Yu, J. Mol. Biol. 50, 509 (1970). C. Lord and N. T. Yu, 1. Mol. Biol. 51, 203 (1970). L. Tomlinson and W. L. Peticolas, J. Chem. Phys. 52, 2154 (1970). Lewis and H. A. Scheraga, Macromolecules 4, 539 (1971).
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on light scattering 2s and fluorescence -°9 in this series should be followed. All chemicals should be of highest available quality. Distilled, degassed water is used, and if solvent fluorescence is apparent further purification may be necessary. ~ Samples may be purified by centrifugation a n d / o r filtration through activated charcoal. Where possible, samples should be filtered through a 0.05-/~m Millipore filter while filling the cell, using 1-ml disposable syringes. Gas bubbles and dust on the inside walls of the cell should be avoided. For measurements in -~H~O, the usual precautions must be followed to avoid contamination by H20. Cacodylate is a useful buffer because it has a very weak Raman spectrum with the exception of a line near 600 cm-1. 26,:" Measurements of Solutions or Suspensions Vertical illumination should be used for clear to moderately turbid solutions or suspensions, with the laser beam focused through a bottom window into the cell, and the scattered radiation collected from the side at 90 ° to the beam. Cells should be made of nonfluorescent fused quartz, with a square or cylindrical cross section, and a flat, well polished bottom. Typical dimensions are 2 mm wide and 15 mm high, but these are not critical. The top of the cell is sealed with a transparent cap to permit deoxygenation and to keep out dust. The cell should be completely filled to avoid background reflections from the curved air-liquid interface. For temperature measurements and for long-term sample stability, the cell should be jacketed or mounted in a massive, temperature-controlled holder. :~° If a jacket is used, the circulating liquid should not pass over the entrance or collection windows. In normal operation, the monochromator collects only the radiation scattered from the cylindrical volume illuminated by the focused laser beam; this is about 2 0 - 5 0 t~m in diameter and 2 - 1 0 mm high. Turbid samples diffuse the beam, as well as the Raman radiation, into a much larger volume so that the number of signal photons which reach the monochromator is greatly reduced. At the same time, noise is increased because of the extensive Tyndall scattering. In such cases we find the following procedure to be effective: The cell is raised so that its bottom is at the midline of the collecting lens and positioned so the laser beam barely grazes the inner surface of the wall closest to the lens. The focusing lens is adjusted so that at the bottom of the cell the beam appears as a bright, sharply defined cylinder, 2-3 mm high and as narrow as possible. The height of the monochromator slit is decreased until the bright image ~"M. Bier, this series, Vol. 4, p. 147. ~ D. J. R. Laurence, this series, Vol. 4, p. 174. ~E. W. Small and W. L. Peticolas, Biopolymers 10, 1377 (1971).
256
CHARACTERIZATIONOF MEMBRANES AND COMPONENTS
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is centered on, but slightly overlaps, the top and bottom of the slit. With care, we have obtained good spectra from suspensions of erythrocyte ghosts (up to 5 m g / m l protein) and B-structured poly-L-lysine (up to 10 m g / m l ) . 6
Differential Spectroscopy In direct measurements of dilute solutions, the broad water background scattering at 1640 cm -1 begins to obscure the solute spectrum at sample concentrations less than 10-20 mg/ml. We have found that the solvent background can be removed by means of differential spectroscopy and that good spectra can be resolved in the Amide I region at concentrations below 1 mg/ml. 6 Our prototype spectrometer uses an oscillating, partitioned cell containing sample and reference solutions in tandem. As the sample (reference) compartments pass alternately through the fixed laser beam, the number of detected photons is added (subtracted from) the memory of a multichannel analyzer, which is synchronized with the cell motion. The spectrometer is null balanced on the strong 3500 cm -1 water band or on an internal reference line in two steps: First, the relative time spent by the laser beam in each compartment during a cycle is mechanically adjusted by shifting the midpoint of the oscillation with respect to the fixed beam. Final balance is through electronic adjustment of the add or subtract periods during which counts are accepted by the analyzer (usually 1 second each). An auxiliary, fixed-wavelength spectrometer monitors the Raman scattering during the course of a measurement and maintains the electronic null, thus compensating for changes in turbidity. This technique allows resolution of defined spectra from structured backgrounds twenty times as large, and is, therefore, capable of separating overlapping bands in complex, multicomponent systems.
interpretation 31 Proteins and Polypeptides. In studying polypeptide solutions, Raman spectroscopy is superior to infrared absorption measurements because it can discriminate between a-helical, B, and random coil conformations. 6,27,~'-'-3' For example, in the Amide I region the three types of struc~1In infrared and Raman spectroscopy the synthetic standards usually employed cannot be taken as absolute or ideal reference materials any more than in optical activity measurements and the experimenter must exercise appropriate caution in the interpretation of data from biological materials. a2M. Smith, A. G. Walton, and J. L. Koenig, Biopolymers 8, 29 (1969). nJ. L. Koenig and P. L. Sutton, Biopolymers 8, 167 (1969); 9, 1229 (1970); 10, 89 (1971). 84p. Sutton and J. L. Koenig, Biopolymers 9, 615 (1970).
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ture can be identified in poly-L-lysine solutions by bands at 1647, 1672, and 1665 cm -1, respectively. 6 Quantitative determination of complex protein structure is difficult, 3~ but it is already apparent that R a m a n spectroscopy can provide at least as much information as can be extracted from optical activity measurements, 27 and is more amenable to theoretical analysis, as well as to kinetic and metabolic applications. While Amide I I scattering is very weak, z*,2~,32-34 the Amide I I I spectrum is readily visible and is more complex than in the infrared. In polypeptides it has been assigned to bands near 1320 cm-1, 2m3-35 and in proteins to a conformation-sensitive region near 1250 cm-1. 24,2~,3~,36 Because of the strength of the S - - S scattering, the disulfide modes are easily observed. 2',3~ The number of bonds is given by the intensity of a line near 509 cm -~, while the ratio of this peak to the C - - S - - S line at 661 cm -1 has been related to the C - - S - - S - - C dihedral angle. 2~,3~
Lipids In aqueous multilayer suspensions of dipamitoyllecithin, the intensity of bands near 1066 and 1130 cm -1 has been taken to a measure of the amount of all-trans structure in the paraffin chains, while a broad band at 1089 cm -1 is assigned to the appearance of structures containing several gauche rotations of the melted paraffin2 ~ The ratios of the 1089:1128 cm -1 or 1089:1066 cm -1 bands provide a sensitive probe of the extent of liquid crystallike order and the cooperativity of the melting transition. F o r the more complex system of the erythrocyte membrane our preliminary studies have shown that P - - O - - C and C z O lipid modes can be resolved together with peptide bands from the protein moiety. Acknowledgments Supported by Grant No. 84750-0 from the U.S. Public Health Service, the Tufts-New England Medical Center Department of Therapeutic Radiology Grant from the National Cancer Institute, and a AFOSR-NRC Fellowship.
~R. C. Lord, Proc. Int. Congr. Pure Appl. Chem. 33(7), 179 (1971). A. M. Bellocq, R. C. Lord, and R. Mendelsohn, Biochim. Biophys. Acta in press. 3, j. L. Lippert and W. L. Peticolas, Proc. Nat. Acad. Sci. U.S. 68, 1572 (1971).