Fourier transform Raman spectroscopy of biological materials

Ira W. Levin and E. Neil Lewis. Anal. Chem. , 1990 ... Lane C. Sander, Matthias Pursch, and Stephen A. Wise. Analytical ... Richard L. Hansen and Joel...
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Laooratory of Cnemical Physics National nstitute of Diaoetes and Digestive and Kianey Diseases National Institutes of Health Bethesda. MD 20892 Since ita discovery more than 60 years ago, the spontaneous Raman effect has enjoyed spectacular success in applications spanning the physical, chemical, biological, and engineering fields. Following the initial excitement generated primarily by physicists in the 19309, Raman spectroscopy was used almost exclusively by chemists for about 20 years for vibrational studies of polyatomic molecules. In the early to middle 1960%visible light lasers as convenient sources of radiation stimulated an enormous amount of interest in Raman spectroscopy. As conventional and resonance-enhanced Raman studies proliferated throughout the 1960s and 1970s, new nonlinear Raman phenomena, distinguished by a plethora of colorful acronyms from CARS t o RIKES. were developed and applied to systems of chemical interest. In 1986, when many Raman spec This article not subjen to US. Copyright Published 1990 American Chemical Society

trow,pists were sensing the serenity of a rapidly maturing field, several investigators ( 1 - 3 ) successfully demonstrated the coupling of Raman-scattered radiatiun t o a Michelson interferometer. replacing the traditional monochroma. tor with a multiplexing instrument. These studies stimulated scientific and economic interest from both the vibrational spectroscopic community and


the manufacturers of FT-IR spectrometers. In the past few years, near-IR FTRaman spectroscopy has evolved as a viable technique for obtaining vibrational Raman spectra of many chemical systems that are either intractable to visible laser excitation or can benefit from the capabilities of interferometric instrumentation (4-9). Although applications to biophysical and biochemical materials have not developed as rapidly as those pertaining to traditional chemical characterizations, FT-Raman spectroscopy holds great promise for providing vibrational data on vast numhers of hiomolecular systems that are not immediately amenable to general Raman and IR techniques. FT-Raman SPmr=.OpY


The advantages of FT-Raman spectroscopy over conventional dispersive Raman techniques, which employ visible laser excitation and photomultiplier detection, include longwavelength excitation to frustrate or eliminate sample fluorescence and improved instrumental performance of FT spectrometers over standard monochromators. The first advantage should not he understated; for


REPORT typical industrial or biological samples, the presence of a dominant fluorescence signal induced by either visible or UV laser radiation often precludes measurement of the weakly scattered Raman light. Because the intensity of the Raman signal is inversely proportional to the fourth power of the excitation wavelength, the near-IR 1064-nm radiation from the NBYAG lasers generally used in IT-Raman instrumentation is less efficient by factors of 16-40 in generating Raman data than,for example, argon-ion sources delivering 514.5-nm emission. In the IT-Raman experiment, this decrease in sensitivity is somewhat compensated for by the interferometer’s intrinsic throughput and multiplex characteristics, referred to as JacquinBt’s and Fellgett’s advantages, respectively. To achieve an effective multiplex advantage, however, we assume that the experiment is detector noise limited, as is the case for the currently available cooled photoconductive germanium (Ge) and indium gallium arsenide (InGaAs) devices. (Although future improvements are expected in detector performance, existing photoconductors now function near their theoretical detection limits). Finally, the capability for precise spectral frequency measurements, which is also implicit in interferometric instrumentation (Connes’ advantage), can influence the decision to use FTRaman equipment rather than scanning, dispersive instruments. For example, in using dispersive instrumentation, one must be sure to maintain the fidelity of the resulting spectral data, particularly after lengthy signal averaging efforts to increase the signalto-noise characteristics of weakly scattering samples. Furthermore, frequency precision is critical in data manipulations involving spectral subtractions. For neat liquids, dilute solutions, or polycrystalline samples, the ability to rapidly record high-quality reproducible Raman spectra with a near-IR NBYAG laser and FT instrumentation is as straightforward as the conventional procedures involving visible laser excitation and dispersive monochromators. Another advantage over dispersive instruments accrues in the use of an interferometer to record moderately high-resolution (0.25 cm-’) Raman spectra. Once a sample is aligned with the entrance aperture of the interferometer in the FT experiment, changes in spectral resolution can be made using onlycomputer keystrokes. (For dispersive instruments, matching the alignment of a sample capillary to an entrance slit corresponding to even 1-cm-I spectral resolution is neither IIOZA

routine nor trivial.) Despite the general advantages and utility of near-IR FT-Raman spectroscopy, the technique has developed more slowly in biology than in chemistry. This more deliberate advancement within the biological disciplines stems from several fundamental problems, including the intrinsic fragility and weakly scattering nature of most native preparations; the ubiquitous and unrelenting fluorescent properties, often representative of biological samples, which can unaccountably affect baseline contours and relative spectral intensities; the difficulties in both monitoring and controlling temperatures of aqueous dispersions; and the rather different aggregation characteristics encountered in intact cellular and model biomolecular systems that make preparation of each new biological sample a separate alignment and focusing effort. The benefits of near-IR laser radiation accrue rapidly when dealing with


biological samples-particularly when persistent fluorescing contaminants are introduced during lengthy sample handling and purification procedures. Because biomolecular samples tend to be fragile,dilute, and weakly scattering aggregates, the intensity of the Raman scattering can be increased dramatically by pelleting the biological suspension directly in the capillary sample tube by ultracentrifugation, a nontrivial exercise. (During centrifugation, the hydrated pellet, which is ultimately illuminated by the laser in the FTRaman experiment, will experience a force of about 150 000-170 OOO g. This necessary sample packing effect can be compared with normal methods for visible laser approaches, in which the pelleting of lipid suspensions requires only a hematocrit centrifuge operating at a force of about 10 OOO 9.) In many cases, the ultracentrifugation step is required not only to record a spectrum effectively,hut is necessary even to observe a signal.

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Disadvantages arise when using near-IR laser radiation with samples dispersed in aqueous media. The overtone region of water absorb radiation from both the Rayleigh signal at the exciting wavelength (9394.5 em-' for the NdYAG laser) and the longer Raman scattered wavelengths corresponding to the C-H stretching modes (6soO cm-' in absolute wavenumbers) of the sample. In addition to the low of Raman scattered intensity for the C-H stretching vibrations, reproducible temperature measurements become a critical concern. In these cases, one resorb to the use of either reference cells or internal temperature calibrations that are based on the spectral sensitivity of the water spectrum. Temperature control can also he maintained by manipulating the incident laser spot size or by using fiber-optic bundles to carry the exciting and scattered radiation in conjunction with sample cooling techniques.

Experimental overview Either a 90' or a 180' backscattering geometry can be used to collect the Raman emission, which is subsequently coupled to a Michelson interferometer. We have used primarily the classic right-angle sampling geometry (Figure 1). initially adopting this approach to accommodate a variety of sampling accessories and optical arrangements (6-9). For samples capable of ahsorbing 1064-nmNdYAG illumination,such

as dilute aqueous biological dispersions, a backscattering geometry (Figure 2) may be more appropriate. Although a single fiber-optic bundle is shown bringing near-IR radiation to the sample in Figure 1,a second fiheroptic collection bundle passing between the sample and the IR source compartment can also be used. In this case, the Raman scattered radiation emanating from the end of the collection fiber replaces the interferometer's IR source, maximizing the coupling of the scattered radiation to the instrumental optics. Removal of both fihers quickly provides the 90O scattering geometry. Whenever even minor changes occur in the arrangement of the optical components or sampling accessories, with respect to the 90' instrumentation, a new phase spectrum must be obtained to ensure accurate spectral representation. Data processing of the enormous numbers of spectra generated is facilitated by using removable disk cartridge systems and a laboratory computer network. The most critical aspects of the FTRaman technique using NdYAG laser excitation lie in efficiently rejecting the extremely intense Rayleigh and reflected signals prior to detection and in choosing suitable, sensitive near-IR detectors. In the 90° system, up to three dielectric notch or narrow bandpass fiters act to discriminate against the Rayleigh scattering, and a solid-state cooled InGaAs detector provides the

Figure 2. Schematic illustrating a 180' backscatteringsample configuration.

necessary detection capability for this region of the spectrum (6).Although efficient Rayleigh line rejection can be obtained by placing the filters perpendicular to the optical axis, an accompanying loss of Raman intensity-particularly for small wavenumber shiftsbecomes evident. By carefully rotating one or two of the filters, one can tune the Raman instrument to record spectra closer to the exciting line. For example, we can obtain spectra down to 100 cm-' (9). To extend the high-frequencylimits of the spectrum, the temperature of the detector can be increased. Increasing the detector temperatwe from -196 'C to -38 "C extends the frequency range from -2990 cm-' to -3300 em-' (9). Under these conditions, we experience only a marginal degradation in the signal-to-noise ratio. A suitable mixing of cold and ambient-temperature nitrogen gas provides a wide range of detector temperatures stable to *1 "C and precludes the neceeaity of constructing separate thermoelectric coolers for the detector element. Rapid improvements will be forthcoming in both fiter and detector technology for extending the high and low frequency limits of the Raman spectrum collected by FT techniques.

Membrane biophysicalsludles Conventionalbilayer assemblies. To understand the conformational and dynamic properties contributing to the complex physiological and pharmacological processes occurring at biological interfaces, a detailed architectural view of cellular membranes at the molecular level is required. For almost two decades, the most productive hypothesis for generating and extending membrane models has been the fluid moaaic construct, in which amphipathic lipids form a bilayer matrix for the expression of a variety of peripheral and integral membrane components (IO). Although most molecular bilayer information pertains to the cellular plasma membrane, the membranes of intracellular organelles exhibit varying properties, functions, and molecular compositions, further demonstrating the richness and complexities intrinsic to biomembranes. In discwing applications designed to elucidate the properties of biomembranes, we will outline several related studies from which both advantages and snares of the FTRaman approach can he seen. Among the many physical tecbniques used to investigate the structural, dynamic, thermodynamic, and functional properties of biomembranes, vibrational spectroscopy provides an extraordinarily sensitive, noninvasive



REPORT meam for probing the many intra- and intermolecular interactions defining the membrane matrix (11). Unlike artificial membranes, biological membrane assemblies that have been subjected to several isolation and purification steps yet remain sufficiently free from fluorescence effecta to allow Raman spectra to be recorded with visible laser excitation are rare. Figure 3 shows the FT-Raman spec-

trum of a bovine synaptic plasma membrane preparation obtained with 1.6 W of near-IR NdYAG laser power at a 4-cm-' spectral resolution. For this sample preparation, which contains integral bilayer proteins and a peripheral protein clathrin coat surrounding the lipid vesicle, it is necessary to pellet the dilute aqueous membrane dispersion received from the cell biologist in the same sample capillary that is ulti-


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Flgun 3. FT-Raman specea of an aqueous dispersion of bovine synaptic mew branes in (a) the 2900-cm-' C-H stretching mode region and @) the 1700650-cm-' spectral region showing attainable frequency preclsionof the spectra.



mately used in the FT-Raman experiment. The membrane is hydrated in an aqueous medium, making heating effects from the 1064-nm laser line substantial. To prevent diffusion of the biomembrane out of the exciting beam and to prevent protein denaturation, the sample is cooled to approximately -40 "C by gently streaming cold nitrogen gas across the capillary. After a two-and-a-half-hour acquisition time, the unsmoothed spectrum (shown in Figure 3) is again representative of an ordered form of a natural membrane. Intact biomembranes often remain liquid crystalline to temperatures below even -20 "C and thus are not routinely examined as a lipid gel phase, as are model systems. The low-temperature phase is specifically characterized by the intensity patterns in the 2845and 2&?80-cm-' acyl chain methylene C-H stretching mode regions and in the 1085- and 1129-cm-I acyl chain C-C stretching mode regions. The frequency values on the spectra illustrate Connes' advantage in the determination of vibrational Raman frequencies with interferometric instrumentation. Because the accuracy of these frequencies depends on measured displacements from the exciting line, the absolute wavenumber of the NdYAG fundamental is measured at the same resolution of the experiment, but without the rejection filters. A precise and accurate measurement of an undistorted lineshape of the exciting fundamental thus can be obtained. If spectra of these dilute, fragile membranes could be recorded dispersively with a scanning monochromator and visible laser excitation, signal averaging times would be so great-even with the intensity advantage of the incident visible radiation-that reliable frequency measurements would probably not be obtainable because of the mechanical limitations of the monochromator. Precise frequency data are important because subtle membrane reorganizations are reflected in frequency shift information; shifts of tenths of a wavenumber are significant, for example, in characterizing gel to liquid crystalline phase transitions. Figure 4 shows the spectra of a model lipid bilayer system consisting of l-palmitoyl-2-oleoylphosphatidylcboline (POPC) and lanosterol in both the gel (a and b) and liquid crystalline (e and d) phases. Lanosterol, a precursor of various sterols in animals, is used as a membrane perturbant. POPC, with one saturated and one unsaturated chain, mimics a general mammalian membrane. The two phases exhibit markedly different intensity and fre-

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quency patterns. Because lanosterol is a bulkier molecule than cholesterol, we have compared temperature-dependent spectra of liposomes reconstituted separately with lanosterol and cholesterol to deduce the modulating effects of sterols on the dynamic and packing properties of the membranes. Interdigitated chain bilayers: lipid/antibiotic interactions. The fluid mosaic model serves as the working hypothesis for modeling membrane behavior, although the initial simplistic view of integral proteins floating in a sea of lipids bas been refined. We now recognize, for example, that proteinprotein contacts within the membrane are common and that the protein influences its surrounding matrix lipids through the formation of annular rings of boundary lipids. Recently, the suggestion and demonstration of explicit domain structures

within the bilayer have begun to provide a basis for further understanding the role of various lipid-lipid and lipid-protein interactions in modifying membrane function. Among the different types of domains available to the bilayer matrix, partially and completely interdigitated bilayer lipid chains are of current biophysical interest (12). In interdigitated chain hilayers, the lipid hydrocarbon chain moieties penetrate across the bilayer center; for completely interdigitated systems, the terminal methyl groups from the hydrophobic region of an opposing monolayer pack near the membrane's more polar interfacial region. In this packing motif, saturated chain diacylphospbolipids, for example, exhibit four hydrocarbon chains per head group instead of the usual two chains per bead group in the conventional bilayer. As a consequence of chain interdigitation, model

membrane systems show a decrease in the thickness of the bilayer and a concomitant ordering of the lipid chains. Consider the interaction of a series of macrolide polyenes, amphotericin B, amphotericin A, and nystatin A, (Figure 5), with a model bilayer composed entirely of the saturated chain diacyl lipid 1.2-dipalmitoylphosphatidylcholine (DPPC). These polyene antibiotics represent a class of natural products with potent antifungal properties. Although it is clear that the mechanism involving antibiotic-cellular interaction occurs at the level of the membrane interface, the nature of the interaction by which cell death is induced by these antibiotics is not fully understood. One common hypothesis suggests that the formation of transmembrane polyene-sterol channels may be involved in allowing cellular components to leak out by increasing the


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Flguro 4. FT-Raman spectra of an aqueous dispersion of 1-palmitoyl-2oleoylphosphatMylchollne(POPC) bilayer assemblies containlng 18 mol % lanosterol in the gel (a and b) and liquid crystalline (c and d) phases. 1106A


membrane's permeability characteristics. Our approach is to examine the morphological response of DPPC bilayers, reconstituted both with and without cholesterol, to the binding activity of amphotericin B and amphotericin A. Figure 6 shows both the conventional dispersive Raman spectrum and the FT-Raman spectrum of the DPPC/amphotericin B complex in the C-H stretching mode region. Because the hydrophobic conjugated polyene portions of the antibiotic ring lead to highly colored yellow-orange compounds, the selective resonance or preresonance enhancement and fluorescent ef-

fects induced by visible and UV laser radiation preclude a complete characterization of the various possible membrane complexes. Thus the upper curve in Figure 6, obtained with argon-ion radiation, is virtually featureless in the important 2900-cm-' spectral region of the DPPC bilayer. In contrast, the near-IR FT-Raman spectrum reflects the bilayer complex in its low-temperature gel phase. The spectral region in Figure 6 is dominated by the two lipid chain methylene features at -2850 and 2880 cm-', which correspond to the CH2 symmetric (in-phase) and antisymmetric (out-of-phase) stretching modes, re-

spectively. In monitoring bilayer responses and membrane perturbations, the peak height intensity ratios I& IZSWand I & I ~ ~ o ,determined as a function of temperature, provide useful indices for gauging lipid packing effects and determining relative bilayer ordeddisorder parameters. In particular, the Im/I2s~ index reflects primarily interchain interactions whereas the IZS~&W intensity ratio measures effects originating from changes in intrachain translgauche isomerization superimposed on the chain-chain interactions (11). Comparisons of the chain-chain interactions between pure multilamellar DPPC assemblies and bilayer complexes of amphotericin A and B, in the presence and absence of cholesterol, are summarized in Figure 7 in terms of the relevant I 2 d I ~ Wintensity parameters. The head and tail of each arrow, representing a specific liposomal assembly or complex, correspond to temperatures of approximately 41.5 "C and 25 "C, respectively. The values of the 12sso/lzssoindices at these two temperatures provide a measure of the intermolecular lipid chain order for the aqueous dispersion of each liposome. (Because of slight differences in the physical antibiotic-lipid packing characteristics, the low gel-phase temperatures may deviate several degrees from the 25 "C DPPC reference bilayer. The possihle small discrepancies in temperature are not problematic, however, because we are concerned primarily with the trends displayed in the figure.) Thus for the pure DPPC multi~ O lamellar system, the I Z S ~ Zpeak height intensity ratio of about 0.77 for the 25 "C gel phase is characteristic of a bilayer whose chains are hexagonally packed. A value of ahout 0.98 reflects a normal liquid crystalline DPPC phase. (The phase transition temperature T, between the gel and liquid crystalline states of aqueous, uncomplexed DPPC dispersions is 41 "C.) Both amphotericin A and B significantly order the bilayer chains (lower values of I=50/12mo); in particular, the 25 OC spectra reflect intensity ratios of 0.67 and 0.65, respectively, for the two complexes. Because other membrane surface-active species (such as polymyxin B, another cyclic antibiotic) are known from X-ray diffraction studies to induce bilayer chain interdigitation, and because the observed Izssa/Izs~ ratios in the 25 "C regime correspond to Raman data for known interdigitated systems, we interpret the lipid-ordering properties of these two polyene macrolide antibiotics as originating from the induced interdigitated chain membrane morphology. In contrast to the




Flgure 6. Dispershre Raman spectrum (top) and FT-Raman spectrum (bottom)of polycrystalline 1.2dipalmitoyiphosphl~lchollne(DPPC) bllayers containing 811). photericin B. (Dwc: Ampholerlcln 0 mole ratlo equals 241.)



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Figure 7. Ordering effects of polyene antiblotlcs on DPPC mullayers. Tlm 1mo/l*Mnimensny ratio rsprewma the I h l " order perameter for hs "arlata IIpoaomeS a1 -25 OC (tall of anow)and 41.6 ' C (M of anow).



spectroscopic and diffraction data derived from model systems, evidence for even partially interdigitated bilayers in native or intact membrane assemblies is limited (23). Despite this paucity of information,. interdigitated chain arrangements may represent a potentially important biological membrane architecture (13,24). As mentioned earlier, membrane cholesterol, an intrabilayer component, is known to modulate the order/ disorder parameters of the lipid matrix. A comparison of the Izs&z8ao order parameters for the various liposomes shown in Figure 7 demonstrates that cholesterol orders the liquid crystalline properties of DPPC multilayers. Although high concentrations of cholesterol generally disorder the gel phase, the lower concentrations of sterol in this experiment (18mol %) exhibit less of an effect. When either amphotericin A or B with 18mol 90cholesterol is added, the I z s s O n z ~ordeddisorder parameters for the membrane chains indicate a gel-phase bilayer ordering that is comparable to systems without cholesterol. If a sterol-antibiotic complex were formed within the bilayer, the gelphase disorder would be suhstantially greater than that reported in Figure 7 because of the disrupting properties of the complex on the lipid chain packing arrangements. Thus, the data suggest that the sterol is expelled from the interior of the bilayer in the low-temperatnre phase; we again speculate about the possibility of interdigitated bilayer chains in these assemblies. Also, referring to Figure 7, we observe that at higher temperatures (41.5 "C) the DPPC-cholesterol-amphotericin A complex is slightly more ordered than the reference DPPC-cbolesterol systems. Surprisingly, the DPPC-cholesterol-amphotericin B complex is as disordered as liquid crystalline DPPC multilayers. It appears that amphotericin A and B, whose conjugated polyene moieties differ slightly, form rather dissimilar cholesterol complexes with the zwitterionic lipid substrate. That is, in the liquid crystalline phase, cholesterol binds externally more strongly to amphotericin B and is removed from the bilayer. In the amphotericin A complex, cholesterol may still remain within the bilayer, ordering the matrix slightly. Antibiotics in the presence of sterol, however, do not have quite the strong bilayer ordering properties observed in the binary lipid-antibiotic systems. (To minimize the differences in preparing the complexes, binary and ternary systems were hydrated from lyophilized mixtures of the appropriate components.)

FlgUrO 8. Near-IR

absorption spectra of HzO and D 2 0 superimposed on the gelphase. near-IR FT-FIaman spectrum of a DPPC bilayer.

Flgure 8. schematic of thermostatted sample assembly for 180° backscattering

geometry. Temperature measurement in aqueoun dispersions. In experiments involving lipid-antibiotic interactions,

the temperatures of the systems were measured with externally placed thermocouples in which the sampling ge-

ometry was calibrated against a known system (such as pure DPPC liposomes) whose temperature behavior haa been well characterized. To exploit fully the FT-Raman methodology in elucidating membrane properties, as in the determination of temperature profiles, it is necessary to measure sample temperatures precisely. The problem of assessing temperatures of aqueous dispersions is illustrated in Figure 8. The absorption spectra of HzO and Dz0 are superimposed on the FT-Raman spectrum of DPPC. Although it is not shown, the exciting NdYAG line at 1064 nm falls in the wings of H20 overtone bands, and the resulting absorption of radiation by the aqueous medium leads to significant sample heating. Increasing the Raman scattered signal by increasing the incident NdYAG power levels can dramatically increase the temperature of the dispersion. (Because the overtone manifold is different for DzO, the heating effect is diminished for D20 dispersions.) In an Hz0 medium, moderate heating effects often lead to a channeling in the sample and subsequently to sample diffusion in the capillary. A h , the Raman scattered C-H stretching mode photons, which are in a particularly important spectral region for interpreting bilayer reorganizations, are absorbed by another HzO overtone, leading to a decrease in the intensity of the observed C-H stretching mode vibrations. Thus one should hydrate model bilayer systems with D20 when possible; most native systems, however, are best spectroscopically surveyed as Hz0 dispersions. For these cases the loss in C-H stretching mode signal can often be tolerated; it is virtually impossible, however, to measure the sample temperature within a desired f0.3 “C error with either externally or internally placed thermocouples. To obviate the temperature measurement difficulties discussed above, an “internal” standard is used. A small reference cell, containing an internal standard (usually D20) is placed in a thermostatted cell aasembly such that the incident NdYAG beam passes first through the reference cell and then through the contiguous sample capillary with its biological dispersion in an H20 medium. Thus the DzO is in intimate thermal contact with the sample canillarv. Figure 9 presents a schematic of the thermostatted cell fabricated for the 180° backscattering geometry. Because the DzO stretching modes reflect a sensitive temperature dependence, and because these Dz0 features occur in the 2500 cm-’ spectral window, library

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curves can easily be generated for determining accurate sample temperatures. (If the sample is dispersed in DzO, no reference cell is required.) A separate DzO reference cell is ideal for investigating lipid-protein complexes, because DzO in the sample capillary would exchange with peripheral membrane protein segments. Using a thermostatted sample chamber and a Peltier device for precise temperature control, we are able to generate library spectra with a dispersive instrument employing visible laser excitation. From data reduction techniques, temperature measurements well within f0.3 OC can be attained from the FTRaman DzO spectra excited with nearIR radiation. Figure 10 shows the differences in measuring the gel to liquid crystalline phase transition region for DPPC using an externally attached thermocouple and the Dz0 reference calibration

method. (In this example the model bilayer assembly was hydrated directly with DzO.) Although fewer temperature points were recorded than are usually required for a complete profile, a temperature-dependent curve in terms of the I z 8 5 0 / Iordddisorder ~~ parameters may still be fit to a two-state model (15) to extract reliable phase transition temperatures T,. The central curves on the figure represent least-squares fits of the spectral data. The outer curves represent the calculated curves plus and minus three standard deviations, respectively. AIthough the gel phase and liquid crystalline phase ordddisorder parameters are correct for the externally placed thermocouple, the highly cooperative, narrow phase transition region characteristic of pure DPPC liposomes is, instead, quite broad, yielding a low T,. For the DzO internal temperature calibration method, however, the correct

I Flgun 11 nperature profile showing the change in lntermolecula~. ..r of pl DPPC multllamallar assemblies using (a) an external thermocouple to determine spectral temperatures and (b) D20 as an Internal temperature callbrant. 11101


DPPC temperature profile is obtained, exhihitingasharp, first-ordergeltoliquid crystalline phase transition at 41.3 “C. The ability to merge under computer control the operations of changing the sample temperature, by means of the solid-state Peltier heat pump, with sample equilibration and spectral acquisition enables us to obtain routinely both ascending and descending temperature profiles, which are subsequently processed through our laboratory computer network. This methodology has been SuccessfuUy applied to a wide variety of model and intact membrane dispersions in studies designed to illuminate the predominant molecular interactions occurring at biological interfaces.

conclusion We have attempted to convey both the flavor of using long-wavelengthexcitation coupled to interferometric instrumentation and the idea that biophysicists and biochemists can provide insight on biomolecular problems that prove unyielding to conventional vibrational spectroscopic approaches. For example, we have obtained near-IR FT-Raman spectra for a wide variety of systems ranging from human and calf lung surfactants to specific proteins related to cataract formation, the expression of Alzheimer’s syndrome, and the insecticidal properties of particular hacilli. In the past our general guide for examining biological samples has been to use dispersive instrumentation if the molecular dispersion was amenable to visible laser excitation with either pbotomultiplier or charge-coupled device detectors. Our techniques have improved to the point that we are now going directly to the near-IR instrumentation to obtain Raman spectra of biomolecules. Despite the somewhat steep learning curve that may be required for handling many biological samples, the flexibility and inherent advantages of near-IR FT-Raman spectroscopy offer sufficient overall benefits to make it the vibrational spectroscopic approach of choice for most biomolecular preparations. For example, the ease of sample alignment with sets of fiber-optic bundles; the interferometer’s multiplex, frequency precision, and resolution advantages; and the ability to control and determine precisely the temperatures of dilute, fragile molecular aggregates all justify an optimistic outlook toward applying FT-Raman spectroscopy to even the more recalcitrant biological systems.

N I H i n 1963. his interests haue ranged from absolute intensity studies and Io9-I4. normal coordinate analyses to eluci(I3) Lewis. E.N.; hvh1. w.;Steer. c. J. Riophys. dating the lipid-lipid and lipid-pro~ Riochim. ~ i ~ ~ kActa ~ 1989. 9 6 161fifi. tein interactions gouerning reorgani(14) Levin, I . W.; Thompson, T. E.; Barenzatiom i n biological membranes. Curh o h y . j H u a n ~ . CRiochemistry . 1985,24. rent interests include aoDlication of ~.~~ .. 62WL*16. (151 Kirchoff. W. H.; Levin, I. w.;J . R ~ ~ I R . and Raman spectroscopies t o charN o t . Bur. Slond. 1987.92,113-28. acterize nouel lipid morphologies. (12) McIntosh. T. J.: Daniel.

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Ira W.Leuin, deputy chief of the Laboratory of Chemical Physics and chief of the section on Molecular Biophysics at the National Institute of Diabetes and Digestive and Kidney Diseases at N I H , receiued a B.S. degree from the Uniuersity of Virginia and a Ph.D. from Brown University. Since joining


E. Neil Lewis receiued a R.S. degree and a Ph.D. from the Polytechnic o f Wales i n the United Kingdom. His research interests include the use of Raman and I R spectroscopies for studying biomembranes and related macromolecular systems of biophysical relevance.


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