Emerging Techniques in Biophysical FT-IR - Analytical Chemistry

Emerging Techniques Richard A. Dluhy National Center for Biomedical Infrared Spectroscopy Battelle Columbus Division Columbus, Ohio 43201

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 (~10~12 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-

in Biophysical FT-IR

Richard Mendelsohn Department of Chemistry Rutgers University Newark, N.J. 07102

Much of our current knowledge of biomolecular 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 1 and 100 Mm (10 2 -10 4 cm - 1 ). 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 biomolecular 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 0003-2700/88/0360-269A/SO1.50/0 © 1988 American Chemical Society

REPORT 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 (2). During the 1960s, 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 advantages 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 · 269 A

structures of photochemical intermedi ates in the ll-cis-retinal chromophore of rhodopsin at 77 Κ (4); isolated active sites of proteins at 4 Κ (5); and, using a relatively new spectroscopic technique, vibrational circular dichroism (VCD), the optical activity of vibrational tran sitions in biomolecules (6). The aim of this REPORT is not to provide a thorough survey of the bio physical IR literature; such a review is beyond the scope of this article. Rath er, we wish to identify several of the emerging spectroscopic techniques and novel applications in biophysical FT-IR that we believe will be increas ingly useful to the biomedical commu nity in the future. ATR of thin biological films Experiments involving oriented sam ples are attractive for biophysical ap plications because they permit the ac quisition of geometric information (orientational order parameters) in addi tion to the normal structural informa tion available from the IR data. A use ful 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 re fractive index of the substrate is much larger than that of the surrounding me dium, the radiation is totally internally reflected. The Fresnel equations indicate that the electromagnetic field penetrates into the rarer medium, even under to tal reflectance conditions. The intensi ty of this evanescent wave falls off ex ponentially 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 exam ple, band intensities and shapes de pend to some extent on the angle of incidence. A detailed discussion of the theory and practice of internal reflection spec troscopy 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 mole cules being studied must be formulated (see, e.g., Fraser [8]). Third, the direc tion of the transition moment with re spect to the axis of molecular orienta tion must be measured or estimated for the given vibrational mode.

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 hemoglo bin with arachidic acid (10). Conforma tional changes in hemoglobin (possibly indicative of /3-pleated sheet secondary structure) were noted upon its interac tion with the lipid. More recently, Kimura et al. report ed spectra of oriented Langmuir-Blodgett films of stearic acid on a germani um plate (11). Examination of the CH2 scissoring region led to the suggestion that the first lipid monolayer has hy drocarbon chains freely rotating about an axis nearly perpendicular to the sur face. 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 lin ear pentadecapeptide antibiotic grami cidin D into multibilayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) ; the initial orientation of the lipids pro duced average tilting angles of 25 ± 7° away from a line normal to the bilayer (12). Insertion of gramicidin had little effect on the orientation of lipid hydro carbon 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 investi gated the conformation and orienta tion of the tetradecapeptide bombesin in films of l-palmitoyl-2-oleoylphosphatidylcholine (POPC) and observed an α-helical conformation with prefer ential orientation normal to the mem brane surface (13). A powerful technique for biochemi cal analysis of oriented monolayer sys tems is the coupling of classical Langmuir-Blodgett transfer techniques with ATR/FT-IR. Briggs et al. ad dressed 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 (14). The E. coli λ phage receptor signal sequence was seen to adopt an α-helical conformation when inserted into the lipid phase. Below the critical insertion pressure of the pep tide, the molecule adopted a β struc ture. A model for the initial steps of the interaction of the signal sequence with biological membranes was postulated. A detailed study of peptide-lipid in teraction was undertaken by Brauner

270 A · 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 pri marily an α-helical secondary struc ture. By considering the peptide to be a bent rigid rod, one could develop a plausible model for the observed orien tation 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 pene trate DPPC bilayers. Surprisingly, the hydrophobic fragment (1-15) pro duced amide I patterns consistent with a structure for a mixture of predomi nantly 0-antiparallel pleated sheet with a smaller fraction of α-helix. In situ spectroscopy of monolayers at the air-water interface Until recently, scientists have lacked a detailed, molecular-level understand ing of the structure and dynamics of interfacial phenomena; most spectro scopic techniques have not provided a way to study flat low-surface-area in terfaces with sufficient sensitivity to produce spectra with reasonable S/N ratios. The IR methods developed dur ing the past decade to study thin mono layer 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 physi cally or chemically adsorbed onto re flective 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 vari ous interfacial phenomena and have re cently been studied as model systems for biological membranes. These monolayer films have been studied us ing ATR techniques; however, the ATR methods require the physical transfer of the monolayers to an appro priate metal substrate (usually via Langmuir-Blodgett techniques) to properly apply the vibrational reflec tance method. Although useful infor mation can be acquired by studying transferred films (see the previous sec tion), one must recognize that the re sulting film is no longer at the A/W

interface, but at an entirely different type of interface, namely, air-metal, (A/M). 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 at 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 at 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 to those of a monolayer at the A/M 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 grazing 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 it 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-pressure/molecular-area phase transition of the DPPC monolayer film at 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-trans hydrocarbon. These data are the first direct spec-

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 LE/LC 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 dichroism

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-

(a) Side view

: : Flat mirrors IR beam

Water

Langmuir-Blodgett trough (b) Top view

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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 the interferometer is brought to a focus at the surface of a custom-designed Langmuir-type film balance through the use of a duallens system.


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Figure 2. The IR external reflection-absorption spectrum of a monolayer of the phospholipid distearoylphosphatidylcholine at the air-water interface. The spectrum plotted above is the result of 4096 scans at 8 c m - 1 resolution. Spectral features can be identified for the C—Η stretching modes of the lipid hydrocarbon chains (~2800-3000 cm" 1 ), the C = Ο stretching vibration of the carbonyl ester (~1740 cm""1), and the P0 2 ~ stretching vibrations (~1250-1100cm" 1 ).

niques. Both are differential spectros copy techniques; spectra obtained rep resent the difference between left and right circular polarization of the in coming light for either the angle of ro tation of the refractive index of the sample (ORD) or the absorption coeffi cients of the sample (CD). During the past decade, the spectro scopic applications of optical activity have been extended into the IR vibra tional region of the spectrum. It has recently become possible to make reli able measurements of the circular dichroism of vibrational transitions, commonly known as VCD. The combi nation of the stereochemical informa tion available from circular dichroism and the local mode information avail able from vibrational IR spectroscopy provides VCD with the potential to de velop into a major new source of solu tion-phase molecular conformational information. VCD was first successfully measured using dispersive IR spectrometers. Measurements worked best in the wavelength range above 2000 cm - 1 . The first VCD spectrum obtained with an FT-IR spectrometer was reported in 1979 (6). The advantages of FT-IR/ VCD over dispersive VCD are the same as those of conventional FT-IR over dispersive IR. In the case of VCD, how ever, these advantages are accentuated by the fact that VCD intensities are 4-5 orders of magnitude smaller than ordi nary IR absorption intensities. The principles of FT-IR/VCD and the ex

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perimental techniques that are neces sary for the measurement of VCD have recently been reviewed (6,18). Researchers have begun to explore the application of VCD measurements to biochemical compounds. To date, most of the VCD work has concentrat ed on amino acids (and their deriva tives) and on small polypeptides or homooligopeptides (see 19-21 and ref erences cited therein). A recent study has also described the VCD in chiral poly (ribonucleic acids) (21). Nafie and co-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 vibrational ly induced electronic ring current mechanism formed by an intramolecu lar hydrogen bond between the C02~ and NH 3 + groups of the amino acid. The implication of this work is that the range of possible solution conforma tions is restricted for these molecules (and perhaps also for simple peptides). The VCD studies of polypeptides have shown that there are characteris tic VCD spectra for the right-handed α-helix, the antiparallel β-sheet, the random coil configuration, and the 3i 0 helix. These structures can be differen tiated on the basis of their unique VCD

signals in the amide I, amide II, and amide A regions. The S/N ratio in the VCD spectra for the solution-phase polypeptides is high enough that Fou rier 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(ribonucleic ac ids) (21). It was found that solutionphase mononucleotides have no signifi cant VCD. However, variation in VCD band shape and magnitude for polynu cleotides 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 in formation concerning biomolecular so lution-phase conformation. Instru mental and theoretical advances, as well as the ability to obtain spectra from single enantiomers, will increase the applicability of this technique for biochemists. Infrared microscopy Although the coupling of an optical mi croscope to an FT-IR spectrometer of fers advantages for biophysical experi ments, the technology has yet to be ex tensively applied in this area. Most reports to date have involved the study of surfaces and polymer inclusions. Re cent investigations in one of our labora tories, however, demonstrated the fea sibility of FT-IR microscopy in a biophysical-biomedical setting. The problem studied was the calcifi cation 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 varia tion in mineral properties. The growth plate is depicted in Figure 3. Samples appropriate for IR transmission spec troscopy were prepared from rat fe murs, which were fixed in formalde hyde and cast into poly(methyl methacrylate) blocks. Thin longitudinal sections (5 μπι) were prepared with a microtome. The poly(methyl methacrylate) was dissolved and the specimen spread on a BaF2 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 fre quency limit at 700 c m - 1 in the experi ment. The spectral region 1200-900 cm - 1 , containing the symmetric and antisymmetric [P02~] stretching modes of the phosphate group from the hydroxyapatite phase, was of interest. Although a detailed analysis of the spectrum will be given elsewhere (22), we note that spectra from various re gions of rat bone (at 20-μπι spatial reso lution) show substantial differences in the structure and relative amounts of

Figure 3. A fairly low-power photomicrograph of the distal femoral growth plate of a normal rat. This section was used to orient an unstained serial section under the FT-IR microscope. Spectra of sites (a), (b), and (c) are shown in Figure 4. The marker at the bottom indicates 250 μιτι. (Photo courtesy of Ed DiCarlo).

mineralizing phase. The ability to extract structural in formation from complex tissues was well demonstrated by the above study. However, several issues must be ad dressed before the technique can be considered generally proven useful in biophysical problems. How is it possi ble to interpret the spectrum of a multicomponent system with highly over lapped bands? Can two-(or three-) di mensional imaging of the data be implemented? How is it possible to ef fectively 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 be lieved would have one dominant com ponent in its spectrum. The expecta

tion was borne out in that the phos phate vibrations of the hydroxyapatite phase could be readily distinguished from other absorbing species. Spectra of multicomponent systems may be an alyzed with correlation spectroscopy, whereas contributions to a spectrum from overlapped bands can be partially sorted out with deconvolution or sec ond derivative spectroscopy. IR imaging in three dimensions (rather than two dimensions plus a constant thickness, as in the work out lined above) is, at best, a doubtful proposition in the mid-IR range. Ex tinction coefficients for water are too high to permit significant penetration of the radiation at a depth greater than 100 μτη. Such limited penetrations would confine the technique to biologi cal structures located near surfaces,


Figure 4. FT-IR spectra from 1800 to 800 cm

1

from three regions of the femur in a normal rat.

The FT-IR samples were made from 5-μιτι longitudinal sections, which previously were fixed in formaldehyde and embedded in poly(methyl methacrylate). Most of the latter was dissolved in methyl acetate prior to FT-IR examination. The section was washed in EtOH and viewed through a BaF2 window. Residual poly(methyl methacrylate) was identified by a band near 1720 cm - 1 . Spectral subtraction was attempted of this feature. Derivative features (a) and (c) indicated shifts in the poly(methyl methacrylate) bands upon interaction with the tissue. Perfect spectral subtraction could not be achieved in these instances, (a) Proliferating zone. Little intensity between 950 and 1150 c m - 1 (compared with the protein bands at 1650 and ~1530 cm - 1 ) indicates that mineralization has not yet commenced, (b) Bottom of the hypertrophic zone. Substantial calcification is occurring, as revealed by the spectral features between 950 and 1100 cm" 1 . The spectrum in this region resembles poorly crystallized hydroxyapatite. (c) Zone of vascular invasion. Extensive calcification is evident from the bands at 950-1100 c m - 1 . Note that the structure of the phosphate band arising from the hydroxyapatite differs from that of Figure 4b, which indicates a different mineral structure. All spectra taken with 4-cm - 1 spectral resolution, 20-μνη spatial resolution, and 200 co-added scans.

where other diagnostic approaches would be more efficient. The near-IR (overtone) region of the spectrum might be better suited for the measure ment—for example, if the spectrum of water in tumor cells differs from that in nonmalignant tissue. The chance of specific identification of particular, mi nor, nonaqueous components is slim. The final issue is that of sampling. The representative sampling of a nonhomogeneous, rapidly varying speci men is a chancy process at best. Solu tions to this problem include decreas ing 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 applications

Photoreceptor pigments. The power and sensitivity of FT-IR in biophysical studies are probably best demonstrat ed 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 de rived from the chromophore. Further more, there is no intensity enhance ment mechanism in IR comparable to resonance Raman scattering. In spite of the technical challenges, several groups have been able to record

changes in the IR spectrum of the chro mophore 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) demon strated the Schiff base in both lightadapted bacteriorhodopsin and in the Κ 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 photocy cle. A year later, Siebert and Mantele used difference IR spectroscopy to in vestigate the transition at 77 Κ of lightadapted bacteriorhodopsin to Κβιο, the first stable low-temperature interme diate (24). They noted a major confor mational change in the Schiff base moi ety immediately after photon absorp tion, with a resultant geometry that differed from those of any of the stud ied model compounds. More recent investigations have cen tered around identification of particu lar amino acids that are altered during the photocycle. Englehard et al. selec tively labeled the protein with 4- 13C aspartic acid (25). A comparison of dif ference spectra in the region of the C02~ stretching vibrations of labeled and unlabeled bacteriorhodopsin indi cated that four aspartic acid residues undergo protonation changes at the Κβιο intermediate. Roepe et al. studied in detail the protonation states of tyro sine and the carboxyl group during the M412 and L550 intermediate (26). They demonstrated the formation of a tyrosinate ion between L550 and M412, as well as alteration in several carboxyl groups between bRsvo and M412 (see Figure 5). It therefore seems possible


that participation of particular amino acids (possibly modified chemically) in protein function may be identified by FT-IR (see, e.g., Roepe et al. [27]). 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-pressure studies. As is the case with most condensed-phase organic molecules, the mid-IR spectra of bio logical components contain broad, highly overlapping band contours, es pecially in the region below 2000 cm" 1 , which contains the greatest amount of structural information. To interpret these complex spectra, most biochemi cal IR spectroscopists perturb their systems in some manner and measure the change in the resulting spectral pa rameters. Temperature profiles, sam ple irradiation, and monolayer com pression have all been employed as en vironmental 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 ex perimental techniques used by bio physical spectroscopists is that of highpressure IR spectroscopy. Although vi brational 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 (maxi mum ~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. Concluding 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 D2O substitution has 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-IR to 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 at 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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BSd Figure 5. FT-IR difference spectra of bacteriorhodopsin at different temperatures and pH's. Spectral acquisition parameters are given in Reference 26. (Adapted with permission from Reference 26.)

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utilizing deconvolution and derivation techniques have shown that these mathematical data-reduction process es can significantly improve the bio chemical spectroscopist's ability to in terpret overlapped IR bandshapes. The application of these resolution-en hancement techniques to biochemical systems was recently reviewed (3). The extreme sensitivity of biomolecular 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 spec troscopy 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 ap plications may be enhanced by the de velopment of VCD and FT-IR micros copy. VCD has the potential to increase our understanding of solution-phase conformation, at least for small biomolecules. For IR microscopy, in formation appears to be obtainable down to the diffraction limit (10-20 μπι). Problems of sampling and spec tral interpretation appear to be tracta ble. Obstacles that once hindered the application of FT-IR to biochemical re search have largely been overcome. As the instrumentation, sampling tech niques, and mathematical algorithms evolve, the application should become more widespread. This report was partially supported by Grant No. RR-01367 (Division of Research Resources, Na tional Institutes of Health), which established the National Center for Biomedical Infrared Spec troscopy.

(11) Kimura, F.; Umemura, J.; Takenaka, T. Langmuir 1986, 2, 96-101. (12) Okamura, E.; Umemura, J.; Takenaka, T. Biochim. Biophys. Acta 1986, 856, 6875. (13) Erne, D.; Schwyzer, R. Biochemistry 1987,26,6316-19. (14) Briggs, M. S.; Cornell, D. G.; Dluhy, R. Α.; Gierasch, L. M. Science 1986, 233, 206-8. (15) Brauner, J. W.; Mendelsohn, R.; Prendergast, F. G. Biochemistry 1987, 26, 8151-57. (16) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373-79. (17) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc, in press. (18) Polavarapu, P. L. In Fourier Trans form Infrared Spectroscopy. Applica tions to Chemical Systems; Ferraro, J. R.; Basile, L. J., Eds.; Academic Press: New York, 1985; Vol. 4, pp. 61-96. (19) Oboodi, M. R.; Brij, Β. Β.; Young, D. Α.; Freedman, T. B.; Nafie, L. A. J. Am. Chem. Soc. 1985,107, 1547-56. (20) Paterlini, M. G.; Freedman. T. B.; Na fie, L. A. Biopolymers 1986, 25, 1751-65. (21) Annamalai, Α.; Keiderling, T. A. J. Am. Chem. Soc. 1987,109, 3125-32. (22) Mendelsohn, R.; Hassankhani, Α.; DiCarlo, E.; Boskey, Α., submitted for publi cation. (23) Bagley, K.; Dollinger, G.; Eisenstein, L.; Singh, A. K.; Simany, L. Proc. Nat. Acad. Sci. USA 1982, 79,4972-76. (24) Siebert, F.; Mantele, W. Eur. J. Biochem. 1983,130, 565-73. (25) Englehard, M.; Gerwert, K.; Hess, B. Kreutz, W.; Siebert, F. Biochemistry 1985, 24, 400-7. (26) Roepe, P.; Ahl, P. L.; Dasgupta, S. K.; Herzfeld, J.; Rothschild, K. J. Biochemis try 1987,26,6696-67Ό7. (27) Roepe, P.; Scherrer, P.; Ahl, P. L.; Das gupta, S. K.; Bogomolni, R. Α.; Herzfeld, J.; Rothschild, K. J. Biochemistry 1987, 26, 6708-17. (28) Siminovitch, D. J.; Wong, P.T.T.; Mantsch, H. H. Biophys. J. 1987,51, 46573. (29) Wong, P.T.T.; Zahab, D. M.; Narang, S. Α.; Sung, W. L. Biochem. Biophys. Res. Commun. 1987,146, 232-38.

References (1) Parker, F. S. Applications of Infrared Spectroscopy in Biochemistry, Biology and Medicine; Plenum: New York, 1971. (2) Griffiths, P. R.; de Haseth, J. A. Fourier Transform Infrared Spectrometry; Wiley-Interscience: New York, 1986. (3) Mantsch, H. H.; Casai, H. L.; Jones, R. N. In Spectroscopy of Biological Sys tems; Clark, R.J.H.; Hester, R. E., Eds.; Wiley and Sons: Chichester, England, 1986, pp. 1-46. (4) Rothschild, K. J.; Marrero, H. Proc. Nat. Acad. Sci. U.S.A. 1982, 79, 4045-49. (5) Fiamingo, F. G.; Altschuld, R. Α.; Moh, P. P.; Alben, J. 0. J. Biol. Chem. 1982, 257,1639. (6) Nafie, L. A. In Advances in Infrared and Raman Spectroscopy, Vol. II; Clark, R.J.M.; Hester, R. E., Eds.; Wiley and Sons: New York, 1984, p. 49. (7) Harrick, N. J. Internal Reflection Spec troscopy; Wiley and Sons: New York, 1967. (8) Fraser, R.D.B. J. Chem. Phys. 1953, 21, 1511-15. (9) Fringeli, U. P.; Gunthard, H. H. In Membrane Spectroscopy; Grell, E., Ed.; Springer-Verlag: Berlin, 1981, pp. 270333 (10) Fromherz, P.; Peters, J.; Muldner, H. G.; Otting, W. Biochim. Biophys. Acta 1972, 274, 644-48.

CIRCLE 63 ON READER SERVICE CARD 278 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988

Richard A. Dluhy (left), a principal research scientist with the Battelle Memorial Institute, has a Ph.D. in chemistry from Rutgers University. His interests include vibrational spec troscopy applied to biophysical sys tems, membrane biophysics, and methods for studying interfaces in situ. Richard Mendelsohn (right), a profes sor of chemistry at the Newark College of Arts and Sciences of Rutgers Uni versity, has a Ph.D. from MIT. His in terests include vibrational spectrosco py and calorimetry applied to prob lems of lipid-protein interaction in membranes.

Emerging Techniques in Biophysical FT-IR - Analytical Chemistry

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