I Rkhard A. Dluhy National Center fa Biomedical infrared Spectroscopy Banelle Coiumbw Division Columbus. Ohio 43201
Rlchard Menddsohn Department of Chemlsby Rutgers University Newark, N.J. 07102
Much of our current knowledge of hiomolecular conformation, structure, and function is derived from spectroscopic methods. These diverse techniques, which include X-ray diffraction, nuclear magnetic resonance (NMR), electron spin resonance (ESR), and optical (visible, UV, fluorescence, and circular dichroism) spectroscopy, are widely used in every active area of biochemical and biophysical research. Many significant. problems, from the determination of three-dimensional structures of proteins and nucleic acids to the development of magnetic resonance imaging (MRI), could only be solved through the application of one or more of these techniques. An additional method, vibrational spectroscopy, has also frequently been employed in the study of biochemical systems. Vibrational (IR and Raman) spectra are observed in the wavelength region between 1and 100 pm (lO*-lO4 cm-I). Physically, IR absorption arises from a changing dipole moment in the molecules during the vibration of interest. The normal modes, in turn, are sensitive to changes in internuclear distances and bond angles, that is, the conformations and configurations adopted by the particular molecular groupings. IR spectroscopy offers several advantages in the study of hiomolecular structure and function. First, unlike UV-vis and fluorescence spectroscopies, which monitor only the chromophores of a target molecule, IR spectroscopy can monitor absorptions in all regions of the biomolecule,because frequencies have been identified that are characteristic of all molecular subgroups. Second, no possibly perturbing probe molecules or synthetic substitutions (such as those required in ESR W03-2700/88/0360-269A/$O 1.5010 @ 1988 American Chemical Society
and some types of fluorescence spectroscopy) are necessary in IR spectroscopy. Third, with the advent of modern Fourier transform infrared (FT-IR) instrumentation, the use of aqueous solvents is no longer proscribed in IR spectroscopy. Therefore physiologically relevant environments, which can be difficult to access with X-ray and neutron diffraction as well as electron spectroscopy for chemical analysis, are readily investigated with IR spectroscopy. Fourth, the time scale of an IR absorption (-lo-'* s) essentially freezes most molecular motions; therefore, the interpretation of experimental results is not complicated by timescale averaging of anisotropic motions as it is with several magnetic resonance techniques. These advantages were recognized by biochemists soon after the develop-
REPOR7 ment of commercial dispersive IR instruments in the 1940s and 1950s. Many early studies concentrated on the empirical correlation of IR spectra with biochemical structure (I). During the 19608, interest in the use of the technique as a biophysical tool waned because of the inherent difficulties of performing the spectroscopic experiments. Five factors contributed to the limited use of IR in biochemistry: the low sensitivity of dispersive IR spectrometers; the intense IR absorption of water, which limited the wavelength regions that could be studied; the limited sampling techniques available for dual-beam dispersive instruments; problems with instrument reproducibility over time; and lack of spectral data-processing techniques for the broad, overlapped bands inherent in condensed-phase IR spectra. The recent resurgence of interest in the application of IR spectroscopy in the study of biochemical systems can be directly traced to the introduction of FT-IR spectrometers during the 1970s. The use of an interferometer rather than a diffraction grating as the mechanical device that encodes wavelength-dependent absorptions results in substantial advantages in the follow-
ing areas: signal-to-noise (S/N) ratio, throughput, speed, and precision. The physical and theoretical bases for these advantqes have been described in the literature (2). One requirement for the development of FT-IR spectrometers was the establishment of a moderately sized computer with considerable storage capacity that could perform the mathematical manipulations involved in the Fourier transform process. Although this observation may seem trivial, the development of data-processing techniques and the routine collection of large amounts of digitized spectra had a profound effect on biophysical IR spectroscopy. As an example, for the first time, the underlying water spectrum could be mathematically subtracted from an aqueous mixture spectrum with great precision, thereby permitting the routine use of water as an IR solvent. Because the spectra of biological molecules are complex and the spectral changes observed from perturbations are usually small, it is often necessary to apply complex data-processing techniques to measure frequencies and bandwidths, or to use sophisticated algorithms such as deconvolution or derivation to the spectrum (3).The availability of digitized data in readily accessible form made these methods possible. The high throughput of FT-IR spectrometers has greatly expanded the range of sampling accessories that can be employed. For example, techniques that demand high sensitivity, such as attenuated total reflectance (ATR) and IR microscopy, are now routinely employed on FT-IR instruments. The new capabilities of IR spectroscopy have presented researchers with broader opportunities and challenges. The role of biophysical FT-IR is unique among the various spectroscopies in use. Rather than duplicate the usual approaches such as NMR, circular dichroism, or fluorescence to study bulk-phase phenomena, research groups are using FT-IR technology to study systems that would be impossible to evaluate using any other method. Investigations using FT-IR include those of very fast time scales and very low temperatures. As examples, the following have been studied using FT-IR
ANALYTiCAL CHEMISTRY. VOL 60. NO. 4, FEBRUARY 15, 1988
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structures of photochemical intermediates in the 11-cis-retinal chromophore of rhodopsin at 77 K ( 4 ) ;isolated active sites of proteins a t 4 K ( 5 ) ;and, using a relatively new spectroscopic technique, vibrational circular dichroism (VCD), the optical activity of vibrational transitions in biomolecules (6). The aim of this REPORT is not to provide a thorough survey of the biophysical IR literature; such a review is beyond the scope of this article. Rather, we wish to identify several of the emerging spectroscopic techniques and novel applications in biophysical FT-IR that we believe will be increasingly useful to the biomedical community in the future.
ATR of thin biological films Experiments involving oriented samples are attractive for biophysical applications because they permit the acquisition of geometric information (orientational order parameters) in addition to the normal structural information available from the IR data. A useful technique for this purpose, one that was popularized by Harrick, is ATR spectroscopy. In this approach, the sample to be studied is cast as a film in contact with a substrate crystal of high refractive index (usually germanium or ZnSe). Source radiation enters through the edge of a trapezoidal prism of the substrate and impinges on the filmsubstrate interface at an angle greater than the critical angle. Because the refractive index of the substrate is much larger than that of the surrounding medium, the radiation is totally internally reflected. The Fresnel equations indicate that the electromagnetic field penetrates into the rarer medium, even under total reflectance conditions. The intensity of this evanescent wave falls off exponentially with vertical distance from the surface and can therefore be used to sample the thin film. The spectral information generated is similar, but not identical, to that available from the normal transmittance mode. For example, band intensities and shapes depend to some extent on the angle of incidence. A detailed discussion of the theory and practice of internal reflection spectroscopy is provided by Harrick (7). In this method, quantitative analysis of the data depends on three factors. First, the Fresnel equations must be solved for the electric field intensities of the particular geometric setup used. Second, a model for the nature of the orientational distributions of the molecules being studied must be formulated (see, e.g., Fraser [8]). Third, the direction of the transition moment with respect to the axis of molecular orientation must be measured or estimated for the given vibrational mode. 270A
The ATR technique is well suited to studies of lipid ordering and lipid-protein interaction in thin films that serve as models for biological membranes. Much information is available about the vibrations of the acyl chains and their transition moment directions (9). An early investigation by Fromherz et al. probed the interaction of hemoglobin with arachidic acid (IO).Conformational changes in hemoglobin (possibly indicative of a-pleated sheet secondary structure) were noted upon its interaction with the lipid. More recently, Kimura et al. reported spectra of oriented Langmuir-Blodgett films of stearic acid on a germanium plate (11).Examination of the CH:! scissoring region led to the suggestion that the first lipid monolayer has hydrocarbon chains freely rotating about an axis nearly perpendicular to the surface. Thicker films showed the next layers of hydrocarbon chains to be aligned at a 30" angle with respect to a line normal to the germanium surface. Okamura et al. incorporated the linear pentadecapeptide antibiotic gramicidin D into multibilayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC); the initial orientation of the lipids produced average tilting angles of 25 f 7' away from a line normal to the bilayer (12). Insertion of gramicidin had little effect on the orientation of lipid hydrocarbon chains, indicating that any gramicidin-induced perturbations were small. Gramicidin D was seen to adopt a helical secondary structure in both DMPC and DPPC films. The axis of the helix was tilted slightly away from the DMPC acyl chains. Erne and Schwyzer recently investigated the conformation and orientation of the tetradecapeptide bombesin in films of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and observed an a-helical conformation with preferential orientation normal to the membrane surface (13). A powerful technique for biochemical analysis of oriented monolayer systems is the coupling of classical Langmuir-Blodgett transfer techniques with ATR/FT-IR. Briggs et al. addressed the problem of interaction of a signal sequence peptide with oriented phospholipids by using LangmuirBlodgett transferred monolayer films, ATR/FT-IR, and circular dichroism techniques ( 1 4 ) . The E. coli X phage receptor signal sequence was seen to adopt an a-helical conformation when inserted into the lipid phase. Below the critical insertion pressure of the peptide, the molecule adopted a 3 structure. A model for the initial steps of the interaction of the signal sequence with biological membranes was postulated. .4detailed study of peptide-lipid interaction was undertaken by Brauner
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
et al., who examined the association of the membrane lytic peptide melittin and two of its fragments with both DPPC and POPC (15).Insertion of the intact peptide into POPC caused large perturbations in acyl chain order, as monitored by the frequencies, halfwidths, and ATR dichroic ratios of the CH2 symmetric stretching modes of the acyl chains. The peptide adopted primarily an @-helical secondary structure. By considering the peptide to be a bent rigid rod, one could develop a plausible model for the observed orientation of the helical segments and for the lytic properties of the peptide. In contrast to the intact peptide, the hydrophilic fragment (residues 16-26) displayed a secondary structure with little helix formation. In addition, this fragment did not significantly penetrate DPPC bilayers. Surprisingly, the hydrophobic fragment (1-15) produced amide I patterns consistent with a structure for a mixture of predominantly $-antiparallel pleated sheet with a smaller fraction of a-helix.
In situ spectroscopy of monolayers at the air-water interface Until recently, scientists have lacked a detailed, molecular-level understanding of the structure and dynamics of interfacial phenomena; most spectroscopic techniques have not provided a way to study flat low-surface-area interfaces with sufficient sensitivity to produce spectra with reasonable S/N ratios. The IR methods developed during the past decade to study thin monolayer films have involved the use of (primarily) transferred films that are studied via reflection techniques. For example, ATR and reflection-absorbance IR spectroscopy have been used to measure the vibrational modes of surface species that have been physically or chemically adsorbed onto reflective substrates, as described in the previous section. One type of interfacial monolayer that has until now proved difficult to study in situ is the class of molecules that form insoluble monomolecular films at the air-water (A/W) interface. These Langmuir-type monolayers were extensively studied as models for various interfacial phenomena and have recently been studied as model systems for biological membranes. These monolayer films have been studied using ATR techniques; however, the ATR methods require the physical transfer of the monolayers to an appropriate metal substrate (usually via Langmuir-Blodgett techniques) to properly apply the vibrational reflectance method. Although useful information can be acquired by studying transferred films (see the previous section). one must recognize that the resulting film is no longer at the A/W
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interface, but a t an entirely different type of interface, namely, air-metal, ( A N ) . From an experimental standpoint, it would be of interest to study these membrane models in situ, under dynamic, interactive conditions. Recently several papers describing the application of external reflection FT-IR to the in situ measurement of lipid monolayers a t the A/W interface have appeared (16, 17). Using the external reflection FT-IR, the IR beam is directed externally to the spectrometer and is specularly reflected from the A/W interface; the water surface acts as the reflective element. The underlying classical electromagnetic theory of monomolecular films a t the A/W interface was analyzed in a recent report (16). When compared with the more traditional situation of a monolayer film at a reflective metal interface, the calculations indicate that the expected intensities of the reflection-absorbance bands for a monolayer at the A/W interface are comparable t o those of a monolayer at the AIM interface. In addition, the following major experimental differences are observed between the external reflectance at the A/W and A / M interfaces: The optimal angle of incidence lies in the range 040' for the A/W interface, unlike the ' incidence reflectance for the
A/M interface; it is possible to obtain spectra for both parallel and perpendicular polarizations for monolayers on water, whereas only parallel polarized spectra are observed on metals; and i t should be possible to observe vibrations in all three geometric orientations, and hence directly deduce molecular conformations for the monolayers on water. These theoretical conclusions were tested in an in situ study of the monolayer phase transition of DPPC (17). This study investigated the liquid-expanded (LE) to liquid-condensed (LC) phase transition of the DPPC monolayer film in order to directly address the questions of conformation and orientation in monomolecular surface films of synthetic phospholipids as a function of surface pressure. The surface-pressurelmolecular-area phase transition of the DPPC monolayer film a t the A/W interface was monitored by observing the frequencies of the CH2 stretching bands. The data showed that at high-molecular areas in the LE phase, the measured frequencies correspond to a highly fluid, disordered lipid acyl chain. Conversely, the measured frequencies of the low-molecular area in the LC phase agree with those of a rigid, mostly all-tram hydrocarbon. These data are the first direct spec-
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troscopic measurements of the molecular conformation in the pressure-area phase transition of a phospholipid monolayer at the A/W interface, and they indicate a continuous change in conformational order throughout the transition. The results show that the DPPC monolayer is heterogeneous and biphasic in character, with coexistence of fluid and solid phases in the L E L C intermediate transition region. Figure 1 illustrates the experimental optical design used in these types of experiments, Figure 2 displays a spectrum of a phospholipid monolayer at the A/W interface. These experiments demonstrate that the external reflection FT-IR technique for studying monolayer films in situ at the A/W interface can reveal unique structural information. The data show that this technique can obtain results of biochemical significance and imply that it can become a mature analytical spectroscopy method with applications in a wide range of surface science problems.
Vibrational circular d i e h r o i Two forms of molecular optical activity well known to biochemical researchers, optical rotatory dispersion (ORD) and circular dichroism (CD), are the bases of widely used spectroscopic tech-
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Figure 1. The optical design for the in situ IR external reflection experiments at the air-water interface. The modulated radiation from me interfernmeter is brougM lo a locus at me Dufsce 01 a curlDmdeslsned Lawmuir-me film balance throvgh the use of a duaC lens system.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15.
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Flgure 2. The IR external reflection-absorption spectrum of a monolayer of the phospholipid dlstearoylphosphatidylchollna at the air-water interface. The specbum plmed above 16 lhe resun of 4096 scans a1 8 cm-' resolution. Specbal lealwes can be
Identified lw lhe C-H ahetching tw$es of me lipld hyjocarbon chaim (-28W-3000 cm-I). lhe C = 0 smching vibration of me carbOnyle81er i-1740 cm-I). and the Po*- suetchima vlhllons (-1250-1100 cm-').
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niques. Both are differential spectroscopy techniques; spectra obtained represent the difference between left and right circular polarization of the incoming light for either the angle of rotation of the refractive index of the sample (ORD) or the absorption coefficients of the sample (CD). During the past decade, the spectroscopic applications of optical activity have been extended into the IR vibrational region of the spectrum. It has recently become possible to make reliable measurements of the circular dichroism of vibrational transitions, commonly known as VCD. The combination of the stereochemical information available from circular dichroism and the local mode information available from vibrational IR spectroscopy provides VCD with the potential to develop into a major new source of solution-phase molecular conformational information. VCD was first successfully measured using dispersive IR spectrometers. Measurements worked best in the wavelength range above 2000 em-'. The first VCD spectrum obtained with an FT-IR spectrometer was reported in 1979 (6).The advantages of FT-IRI VCD over dispersive VCD are the same as those of conventional FT-IR over dispersive IR. In the case of VCD, however, these advantages are accentuated by the fact that VCD intensities are 4-5 orders of magnitude smaller than ordinary IR absorption intensities. The principles of FT-IRNCD and the ex-
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15. 1988
perimental techniques that are necessary for the measurement of VCD have recently been reviewed (6,18). Researchers have begun to explore the application of VCD measurements to biochemical compounds. T o date, most of the VCD work has concentrated on amino acids (and their derivatives) and on small polypeptides or homooligopeptides (see 19-21 and references cited therein). A recent study has also described the VCD in chiral poly(rihonuc1eic acids) (21). Nafie and eo-workers examined the C-H stretching region of the majority of naturally occurring amino acids and a number of amino acid transitionmetal complexes. They proposed a chirality rule for the methine C,-H stretching mode in these molecules based on the observation of a strong, monosignate positive VCD intensity. In addition, they postulated that the origin of this VCD bias is a vibrationally induced electronic ring current mechanism formed by an intramolecular hydrogen bond between the COzand NH3+ groups of the amino acid. The implication of this work is that the range of possible solution conformations is restricted for these molecules (and perhaps also for simple peptides). The VCD studies of polypeptides have shown that there are characteristic VCD spectra for the right-handed u-helix, the antiparallel p-sheet, the random coil configuration, and the 3,,,helix. These structures can be differentiated on the basis of their unique VCD
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signals in the amide I, amide 11, and amide A regions. The S/N ratio in the VCD spectra for the solution-phase polypeptides is high enough that Fourier self-deconvolution techniques can be applied to the VCD spectra. The VCD technique also has recently been applied to chiral phosphate biomolecules such as poly(ribonuc1eic acids) (21). I t was found that solutionphase mononucleotides have no significant VCD. However, variation in VCD hand shape and magnitude for polynucleotides can be correlated with base stacking, base pairing, and degree of order. The results to date indicate that VCD has the potential to increase the amount of available experimental information concerning biomolecular solution-phase conformation. Instrumental and theoretical advances, as well 88 the ability to obtain spectra from single enantiomers, will increase the applicability of this technique for biochemists.
Infrared mlcrascopy Although the coupling of an optical microscope to an FT-IR spectrometer offers advantages for biophysical experiments, the technology has yet to he extensively applied in this area. Most reports to date have involved the study of surfaces and polymer inclusions. Recent investigation~in one of our laboratories, however, demonstrated the feasibility of FT-IR microscopy in a biophysical-biomedical setting. The problem studied was the calcification in the growth plate of normal rats and those deficient in vitamin D. Mineralization occurs across a narrow zone of transition (1-2 mm), providing a useful site for studying spatial variation in mineral properties. The growth plate is depicted in Figure 3. Samples appropriate for IR transmission spectroscopy were prepared from rat femum, which were fixed in formaldehyde and cast into poly(methy1 methacrylate) blocks. Thin longitudinal sections (5 pm) were prepared with a microtome. The poly(methy1 methacrylate) was dissolved and the specimen spread on a BaFz window. FT-IR spectra from various regions are shown in Figure 4. Cut-off of the narrow-band mercury cadmium telluride detector set the usable lower frequency limit at 700 em-' in the experiment. The spectral region 1200-900 em-', containing the symmetric and antisymmetric [PO%-] stretching modes of the phosphate group from the hydroxyapatite phase, was of interest. Although a detailed analysis of the spectrum will he given elsewhere (22), we note that spectra from various regions of rat bone (at 20-pm spatial resolution) show substantial differences in the structure and relative amounts of
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Figure 3. A fairly low-power photomicrograph of the distal femoral growth plate of a normal rat. This section WBS usad to oriem an ~ S t a l n e dserlal section under the FT-IR microscope.Spectra of sites (a). (b), and (c) are Shown in Figure 4. me marker at the bottom indicates 250 am. (Photo counesy of Ed DiC~rlo).
mineralizing phase. The ability to extract structural information from complex tissues was well demonstrated by the above study. However, several issues must be addressed before the technique can he considered generally proven useful in biophysical problems. How is it possible to interpret the spectrum of a multicomponent system with highly overlapped bands? Can two-(or three.) dimensional imaging of the data be implemented? How is it possible to effectively sample a biological specimen that may have rapid spatial variation in the concentration of various species and hence in its FT-IR spectrum? In the experiments discussed above, researchers selected a sample they believed would have one dominant component in its spectrum. The expecta-
tion was borne out in that the phosphate vibrations of the hydroxyapatite phase could be readily distinguished from other absorbing species. Spectra of multicomponent systems may be analyzed with correlation spectroscopy, whereas contributions to a spectrum from overlapped bands can he partially sorted out with deconvolution or second derivative spectroscopy. IR imaging in three dimensions (rather than two dimensions plus a constant thickness, as in the work outlined above) is, at best, a doubtful proposition in the mid-IR range. Extinction coefficients for water are too high to permit significant penetration of the radiation a t a depth greater than 100 rm. Such limited penetrations would confine the technique to biological structures located near surfaces,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4. FEBRUARY 15, 1988
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FT-IR spectra from 1 8 L JOO cn. ..nn three regions of the femur in a normal rat. The FT-iR pampie w e rade horn 5-pm iongiludinaiseclions. Vmich previously w m fixed in fcimak%hyde and embedded in poiy(methyi meU~uy!ata).b t of mS ianer wa8 dissolved in nmmyl acetate p i a to FT-iR eminauon. The section was washed in E m and viewed through a @aFIwlndow. Residual Poiy(memYi Kmmaayiata) was identifiedby a band near 1720 cm-'. Spectral subb'aclion WBS anempted 01 this featwe. Derivafi features (a) and (c) indicatedshim in me paMmihyimsthacryiata)bands upan interactionwim me ti-. P M spectral subbactim wuid not be achieved in mess instances. (a) froiiferating m e . LMie intenaw M e e n 950 and 1150 an-' (cornwedwith me protein bar& at 1050 and -1530 an-') indicates mat mineraiiration has ml yet wmrrmnced. (b) BOltOm of mS hypertmphic-. Sub618nliai aicificetion is occuring. 86 revealed by me specbal features beween 950 and 1100 an-'. The specbum in mis rwim ,-&bas poorly uyataliizedhydonlapatite.(c) zone of yadcuiar invasion. ExtensiM ceicifimtion is evident from the bands at 950-1 100 cm-'. Note hat me sbuchns of the phosphate band wising hom me hydroxyapatite differs horn mal ot Fig- 4b. which indicates a different mineral sbuctus. Ail spectra taken wHh Ccm-' specbni resoiution. 2&pm spatial resoiution, and 200 wdded scans.
where other diagnostic approaches would be more efficient. The near-IR (overtone) region of the spectrum might be better suited for the measurement-for example, if the spectrum of water in tumor cells differs from that in nonmalignant tissue. The chance of specific identification of particular, minor, nonaqueous components is slim. The final issue is that of sampling. The representative sampling of a nonhomogeneous, rapidly varying specimen is a chancy process at best. Solutions to this problem include decreasing spatial resolution (to larger diameters of field) and sequentially sampling overlapped spatial regions. Some type of spatial transform could then be used to monitor relative amounts of material in particular (twoor three-dimensional) domains.
Novel appUcations Photoreceptor pigments. The power and sensitivity of FT-IR in biophysical studies are probably best demonstrated by recent investigations of bacteriorhodopsin, a light-driven proton pump isolated from halophilic bacteria. This protein has been widely investigated as the archetype for intrinsic membrane proteins. It is of interest to measure changes that occur in the vicinity of the retinal chromophore during the photocycle that this molecule undergoes. The experiment requires the high S/N ratios achievable with FT-IR, because less than 1%of protein weight is derived from the chromophore. Furthermore, there is no intensity enhancement mechanism in IR comparable to resonance Raman scattering. In spite of the technical challenges, several groups have been able to record 276A
changes in the IR spectrum of the chromophore or in particular amino acids in the protein that occur during its proton pumping cycle. Five years ago, Bagley et al. (23)and Rothschild and Marrero ( 4 ) demonstrated the Schiff base in both lightadapted bacteriorhodopsin and in the K intermediate to be protonated. In these studies, difference spectroscopy was used to isolate those vibrations that changed in intensity or frequency between various states of the photocycle. A year later, Siehert and Mantele used difference IR spectroscopy to investigate the transition at 77 K of lightadapted bacteriorhodopsin to K610. the first stable low-temperature intermediate (24).They noted a major conformational change in the Schiff base moiety immediately after photon absorption, with a resultant geometry that differed from those of any of the studied model compounds. More recent investigations have centered around identification of particular amino acids that are altered during the photocycle. Englehard et al. selectively labeled the protein with 4- 13C aspartic acid (25).A comparison of difference spectra in the region of the COz- stretching vibrations of labeled and unlabeled bacteriorhodopsin indicated that four aspartic acid residues undergo protonation changes at the K610 intermediate. Roepe et al. studied in detail the protonation states of tyrosine and the carboxyl group during the Malz and LSmintermediate (26). They demonstrated the formation of a tyrosinate ion between L5m and Mal*. as well as alteration in several carboxyl groups between bRsTo and Mal? (see Figure 5). It therefore seems possible
ANALYTiCAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
that participation of particular amino acids (possibly modified chemically) in protein function may be identified by FT-IR (see, e.g., Roepe et al. 1271). In addition, the nature of the structural changes undergone by these particular amino acids may be determined from the nature of the alterations in the FTIR spectral parameters. High-preseure studies. As is the case with most condensed-phase organic molecules, the mid-IR spectra of biological components contain broad, highly overlapping band contours, especially in the region below ZOO0 cm-I , which contains the greatest amount of structural information. To interpret these complex Epectra, most biochemical IR spectroscopists perturb their systems in some manner and measure the change in the resulting spectral parameters. Temperature profiles, s a n ple irradiation, and monolayer compression have all been employed as environmental variables. IR parameters such as frequencies, bandwidths, and intensities can be measured directly from the resulting spectra or obtained from difference spectra. A recent addition to the range of experimental techniques used by biophysical spectroscopists is that of highpressure IR spectroscopy. Although vibrational spectroscopists have for some time used the experimental technique of the variation of hydrostatic pressure within the gaskets of a diamond anvil cell, they have only recently begun to explore its use as a probe of biophysical systems. Wong and co-workers applied highpressure FT-IR spectroscopy to the study of the pressure-induced (maximum -50 kbar) structural phase tran-
sitions in the hydrocarbon chains of a series of phosphatidylcholine molecules (see 28 and references cited therein). They showed that the barotropic behavior of these molecules can be used as an indication of intrachain conformational and interchain orientational processes. They also showed that the extent of interdigitation in the acyl chains of the two opposing phospholipid monolayers in the membrane bilayer can be monitored using this technique. The pressure-dependent (maximum -38 kbar) IR spectra of live bacterial strains of E. coli were measured in a recent study (29). It was shown that FT-IR could distinguish those strains of E. coli that had been genetically altered to overproduce recombinant proinsulin. The implication of this study is that the barotropic behavior of the bacteria’s IR spectra can be used to monitor gene expression in microorganisms.
concludhg remarks Scientists are beginning to recognize the power of the current generation of FT-IR instruments in biophysical studies. The advantages of the sampling requirements (e.g., small amounts of material, no probe molecules necessary, many physical states accessible) enhance the attractiveness of the method. Problems with the strong IR absorption of water have been somewhat alleviated, although not completely eliminated, through use of ATR, difference spectra, and derivative spectroscopy. Although DzOsubstitution bas sometimes been used to enhance the observability of certain spectral regions, it must be remem-
bered that H-D exchange alters the frequencies of protein peptide bond modes. This phenomenon has been used to investigate the accessibility of various types of secondary structure to the exchange process, but on the whole it is a nuisance that increases the difficulties of spectral analysis. Although the experimental acquisition of biochemical FT-IR spectra generally remains a difficult task, the nature of the information available from FT-IR studies of biomolecular structure and organization is possibly of a broader scope than is generally thought. Traditionally the method has been used mostly to monitor protein secondary structure. However, the ability of reflectance techniques to measure the orientation of particular secondary structures has added a new dimension to the study of protein and peptide insertion in biological membranes. Especially powerful is the ability of FT-IRto sample oriented monolayer films of biomembrane components, either by transferring these films onto solid supports (i.e., ATR crystals) through Langmuir-Blodgett techniques, or by using external reflection spectroscopy to directly examine these monomolecular films a t the A/W interface. Researchers are just beginning to exploit these new approaches to obtain detailed molecular-level information regarding membrane interfacial properties. One of the largest drawbacks to the routine use of FT-IR in the biomedical sciences continues to be the inherently broad bandwidths and multiple overlapped band contours in solutionphase IR spectra. Recent advances in
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Figure 5. FT-IR difference spectra of bacteriorhodopsln at different temperatures and pHs. Spectral acquisition parameters are given in Reference 26. (Adapted with permisdon I r a ReIBrence 28 )
1
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utilizing deconvolution and derivation techniques have shown t h a t these mathematical data-reduction processes can significantly improve the hiochemical spectroscopist's ability to interpret overlapped IR handshapes. The application of these resolution-enhancement techniques to biochemical systems was recently reviewed (3). The extreme sensitivity of hiomoleculm FT-IR spectroscopy was recently demonstrated in several studies of bacteriorhodopsin (26, 27). In this case, structural alterations at particular sites (chromophores or amino acids) in proteins were observed and their role in function evaluated. When FT-IR spectroscopy has been coupled with the methods of genetic engineering or chemical modification, a powerful tool will have been implemented. The role of FT-IR in biomedical applications may he enhanced by the development of VCD and FT-IR microscopy. VCD has the potential to increase our understanding of solution-phase conformation, at least for small biomolecules. For IR microscopy, information appears t o be obtainable down to the diffraction limit (10-20 pm). Problems of sampling and spectral interpretation appear to be tractable. Obstacles that once hindered the application of FT-IR t o biochemical research have largely been overcome. As the instrumentation, sampling techniques, and mathematical algorithms evolve, the application should become more widespread.
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This report was partially supported by Grant No. RR-01367(Division of Research Resources, National Institutesof Health), which established the National Center for Biomedical Infrared Spee-
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R.J.M.; Hester, R. E., Eds.; Wiley and Sons: New York, 1984,p. 49. (7) Harriek, N. J. Internal ReflectianSpectioseopy: Wiley and Sons: New York,
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form Infraied Spectroscopy. Applications to Chemical Systems; Ferrsro, J. R.; Basile, L. J., Eds.; Academic Press: New York, 1985;Vol. 4,pp. 61-96. (19) Oboodi, M. R.; Brij, B. B.; Young, D.A.;Freedman,T.B.;Nafie,L.A.J.Am. Chem. Soc. 1985.107.1547-56. (20) Paterlini, M. G.; Freedman. T. B.; Nafie, L. A. Biopolymers 1986,25,175145. (21) Annamalai, A,; Keiderling, T. A. J. Am. Chem. Soe. 1987,109,3125-32. (22) Mendelsohn, R.; Hassankhani, A,; DiCarlo, E.; Boskey, A., submitted for publi-
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(23) Bagley, K.; Dollinger, G.; Eisenstein, L.; Sing?, A. K.; Simany, L. Proc. Not. Aead. Sa. USA 1982,79,4972-76. (24) Siebert. F.: Mantele. W. Eur. J. Bioehem. 1983,130,565-73. (25) Englehard, M.; Gerwert, K.; Hem, B.
Kreutz, W.; Siebert, F. Emchemistry
1985,24.400-7. (26) Roepe. P.;Ahl, P. L.;Dssgupta, S. K.;
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try 1987,26,66966707. (27) Roepe, P.;Scherrer, P.; Ahl, P. L.; Dasgupta, S. K:; Bogamolni, R. A.; Herzfeld, J.; Rothsehild, K. J. Biochemistry 1987, 26,6708-17. (28) Siminovitch, D. J.; Wong, P.T.T.; Mantsch, H. H. Biophys. J . 1987,51,46571 , ,.
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For Amlication
Other light-sensitive instruments
Umemura, J.; Takenaka,
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troseopy.
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Color and SO, Analyzers
(11) Kimura, F.;
CHEMISTRY, VOL. 60. NO. 4. FEBRUARY
15, 1988
Richard A . Dluhy (left), a principal research scientist with t h e Battelle Memorial Institute, has a Ph.D. i n chemistry from Rutgers University. His interests include uibrational spectroscopy applied t o biophysical syst e m s , membrane biophysics, a n d methods for studying interfaces in situ. Richard Mendelsohn (right), a professor of chemistry a t the Newark College of Arts and Sciences of Rutgers University, has a Ph.D. f r o m MIT. His interests include vibrational spectroscop y and calorimetry applied t o problems of lipid-protein interaction in membranes.
