Department of Biochemistry, New Yorlc Medical College, Basic Science Building, Valhalla ... steroids, porphyrins, enzymology, and medical uses, among others.
feature article
Biochemical Applications of Infrared and Raman Spectroscopy Frank S. Parker Department of Biochemistry, New Yorlc Medical College, Basic Science Building, Valhalla, New Yorl~ 10595 (Received 25 June 1974; revision received 9 October 1974) Uses to which infrared and Raman spectroscopy have been put in biochemical work are reviewed. In the infrared field some of the applications discussed are hydrogen bonding, carbohydrates, lipids, proteins and polypeptides, hydrogen-deuterium exchange, nucleic acids, steroids, porphyrins, enzymology, and medical uses, among others. In the Raman field some of the applications discussed are polypeptides, proteins and enzymes, nucleic acids, carbohydrates, steroids, membranes, lipids, and calcified tissues. Some applications of resonanceRaman spectroscopy are discussed. They include such substances as carotenoids and plant pigments, enzymes, hemoglobin, cytochrome c, hemocyanin, visual pigments, and proteinligand and antibody-hapten interactions. The kinds of information obtainable with each of the methods and the various classes of biochemical substances are given in some detail, and pertinent references are supplied. INDEX HEADINaS: Infrared spectroscopy; Raman spectroscopy; Infrared, general; Infrared, spectra; Methods, analytical; Molecular structure.
INTRODUCTION It is timely to have a review paper concerning biochenfical applications of infrared and Raman spectroscopy because, although both fields of spectroscopy are well known to our reading audience, many are probably unaware of the contributions that these disciplines can make and have made to solving biochemical problems. The advances in applying Raman spectroscopy are being published at a rapid pace. New infrared spectroscopic applications are still coming out, although not so profusely. Both methods remain important tools for the investigation of organic and biochemical molecules. We hope that this paper will give our readers an adequate representative sampling of applications from the literature and will render an interesting overview. Infrared spectroscopy has been used very frequently for about 20 years for studies of biological molecules and their reactions. The reader will find hundreds of studies mentioned in Refs. 1 to 3. Raman spectroscopy has also been applied to biochemical systems for many years, but not nearly so frequently because of instrumental limitations until recently. Edsall4 studied the ionization of amino acids and related compounds by Raman spectroscopy as early as 1936. He and his coworkers studied such biological molecules in aqueous solution, 5-9 namely, the effects of ionization and of deuterium substitution. Garfinkel and Edsall7 recorded the spectrum of the enzyme lysozyme. All the experimentation of Edsall et al. used mercury arc excitation and photographic recording. Work in the field of Raman Volume 29, Number 2, 1975
spectroscopy has increased markedly with the advent of laser excitation. Much interesting material is appearing in the literature now concerning the use of resonance-Raman spectroscopy. We mention below a few examples of its use biochemically. We assume that the reader is generally familiar with the theory of infrared and Raman spectroscopy and the instrumentation used in these fields of investigation. However, a partial listing of references is given here. 1°-21
I. INFRARED SPECTROSCOPY A. Sampling Methods 1. Solids Many of the techniques used for the preparation of samples have been described in detail in several sources.l-3, 12.1~, 15, 17One can readily record the spectra of many solid compounds as mulls and as alkali halide pellets, or without the use of a suspending medium, as in films, or by means of single or multiple internal reflection?~ Each of these techniques has its usefulness and advantages, but one must be aware of the disadvantages also 1: for example, the interference from C--H stretching bands of mineral oil; the fact that some compounds exist in polymorphic forms and show differences in infrared spectra; the fact that certain organic compounds interact with the alkali halide of the pellet; or the fact that transmission and internal reflection data do not always agree. A very useful technique for preparing a mixture of a biochemical substance and alkali halide is to freeze APPLIED SPECTROSCOPY
129
rapidly an aqueous solution of the mixture and remove the water under vacuum (lyophilization). This method is frequently used to advantage in biochemistry laboratories. Solid films have been studied frequently in biochemical work, for example, in structural studies of proteins, polypeptides, and polysaccharides. Such films have been of particular value for studying polarization spectra of macromolecules in intact films and in oriented ones (stretched, rolled, or stroked), thereby permitting knowledge to be gained about spatial arrangements and conformational effects in the molecules. The use of polarized infrared radiation and the measurement of dichroism have been discussed in detail in Refs. 1 and 15. Proteins and polypeptides have been studied in detail by means of polarized infrared radiation and their various types of structure have been distinguished (see Chap. 10 in Ref. 1 and Ref. 23), for example, a-helix, parallel-chain pleated sheet, and antiparallel-chain pleated sheet. An example of the use of this technique for studying a stretched film of sodium hyaluronate, a mueopolysaeeharide, has been given by Quinn and Bettelheim. 24 An interesting study of human hair keratin was done by Baddiel, 2~ who used the attenuated total reflection technique. His data suggested that the hair cuticle has a mixed protein configuration composed largely of the a-helix with contributions from the/3 or extended form, and the central cortical material is mainly a mixture of a-helix and random coil or amorphous form. Katon et al. ~6 used low temperature techniques to study biochemical substances. For example, they showed the spectra of lactose monohydrate at three temperatures--298, 113, and ~ 2 0 ° K (temperature of boiling hydrogen)--and found that the spectrum at 20°K has the best resolution and the greatest band intensities. The spectra also show that, on cooling of the sample, a bonded-OH band breaks up and shows much fine structure. Katon and his co-workers have also reported the effects of low temperature on other carbohydrates and different types of compounds, for example, cholesterol, urea, and carnosine.~ Hermann et al. ~8 have reviewed the subject of infrared spectroscopy at subambient temperatures.
The transparency of the deuterated solvents in certain regions allows them to be used for qualitative and quantitative analyses of dissolved solutes where absorption bands are obscured by nondeuterated solvents. Mieromethods have been described 1, ~, 13 for application to solutions and solids. These methods are important because frequently biochemical samples are minute. B. Applications
In a paper of this length we are limited to discussion of a few examples to demonstrate the applications of infrared spectroscopy to a variety of molecules and the processes affecting them. 1. Hydrogen Bonding
Comprehensive coverage of the subject of hydrogen bonding is given in books by Pimentcl and McClellan 32 and Hamilton and Ibers. ~3 Hydrogen bonding in biological systems, in particular, has been discussed by EngeP ~ and LSwdin2 a (See also the index in Ref. 1.) The self-association of cholesterol and its interaction with triglycerides by hydrogen bonding has been studied.i, 86 The dimerization constant Kd for the selfassociation of cholesterol in CC14 was calculated from infrared measurements of the OH-stretching band of cholesterol (Fig. 1) and was found to be 4.5 liters/mole at 23°C. The enthalpy of dimerization, - 1.8 kcal/mole, was determined from the dimerization constants measured at different temperatures between 5 and 50°C. Infrared spectra of mixed solutions of cholesterol and triacetin, tributyrin, or trilaurin gave evidence of the formation of a 1:1 hydrogen-bonded complex. The equilibrium constants and enthalpies of formation of
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The use of solvents for infrared spectroscopy has been described often enough in many textbooks. 1, 12, 13.15,59.30 In the case of many organic biomolecules the usual range of solvents is adequate, for example, carbon tetrachloride, carbon disulfide, chloroform, etc.; but for many of the very important classes of biological molecules only water or D20 will suffice. Proteins, nucleic acids, mucopolysaccharides, lipoproteins, and other such complex compounds in their natural habitat either are dissolved in water or are in very close contact with it. We shall have more to say on this topic later when we discuss various kinds of biological molecules. McNiven and Court 8~ have published the spectra of 12 solvents in their deuterated and undeuterated forms. 130
Volume 29, Number 2, 1975
I,z [z: bJ CL
37 o
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FIG. 1. OH-stretching vibration bands of cholesterol at different concentrations. Solvent, CC14; temperature, 23°C. The ordinate axis is not drawn to scale. Reprinted with permission from Ref. 36. Copyright by the American Chemical Society.
the complexes of cholesterol with each of the triglycerides were calculated. The K values at 23°C ranged from 2.4 to 3.7 liters/mole, and the AH values from - 3 . 5 to - 5 . 4 kcal/mole. Cole and Macritchie 37 used intensity measurements of the OH-stretching band to determine the energy of the hydrogen bond formed between a substituted cyclohexanol and dioxane in tetrachloroethylene solutions. They found a value of 3.2 ~ 0.3 kcal/mole for the hydrogen bond in the trans-dihydrocryptol-dioxane system. 2. Carbohydrates
Many details of carbohydrate spectra and correlated structures have been given in Ref. 1. These refer, for example, to positions of various types of infrared bands of D-glucopyranose derivatives, as of D- or L-aldopyranoses and their derivatives, 38, 39 and to anomerdifferentiating bands of methyl pyranosides,~° namely, D-xylo and L-arabino forms. OrP 1 made some assignments for the chondroitin sulfates. Since polysulfated hyaluronic acid, which has equatorial sulfate groups only, absorbs at 820 cm-1, he concluded that the sulfate group of chondroitin sulfate C showing a band at 825 cm -~ is equatorially attached and that the sulfate group of chondroitin sulfate A showing a band at 855 cm-~ is axially attached. He ascribed the bands to the C--O--S vibration. Lloyd and Dodgson42 later found that the equatorial sulfate group in D-glucose 3-sulfate has a band at 832 cm-1, and that a band at 820 cm-1 is displayed by the 6-sulfates of D-galactose, D-glucose, and 2-acetamido-2-deoxy-Dglucose, in which the ester group is on the equatorial primary hydroxyl group. Therefore, chondroitin sulfates C and D probably have an equatorial sulfate group on C-6, and chondroitin sulfates A and B have an axial sulfate on C-4 of the 2-acetamido-2-deoxy-D-galactose residues (see Fig. 2). (Note: Turvey et al., 43 Peat et al., ~4 and Harris and Turvey 45 have commented on the dangers inherent in using only infrared data to assign positions to sulfuric ester groups on sugar rings and to sulfonic esters of pyranose derivatives.) The mutarotation of a-D-glucose, ~-D-glucose, and /~-D-mannose has been measured 46 and the kinetics have been calculated from infrared measurements. In a paper mentioned earlier 24 sodium hyaluronate
isolated from umbilical cords was studied by polarized infrared radiation. The authors were able to assign bands to various structural features of the molecule; for example, 1330 cm-1 was assigned to amide III, 1625 cm-1 to CO of the N-acetylamine group, and 1155 cm-~ to antisymmetric bridge-oxygen stretching. 3. Lipids O'ConnoP 7 has presented a chart of absorption bands employed in the applications of infrared spectroscopy to fatty acid chemistry. Many other applications have been cited 1 concerning lipid research in general. The occurrence of organophosphonic acids and their derivatives (Fig. 3) has been reported in a wide variety of biological material. One example of work in this area is that of Baer and Stanacev, 4s who synthesized phosphonic acid analogs of lecithins which were usable as reference compounds in the elucidation of the structure and configuration of naturally occurring phosphonolipids and as valuable substrates for the study of enzymatic reactions. Fig. 4 shows a lecithin structure. The infrared spectra of lecithins and their phosphonic acid analogs showed several differences in the region of 1333 to 833 cm-1, thus differing from cephalins and their phosphonic acid analogs whose spectra are almost identical, except for one band. 49 The phosphonie acid analogs of lecithins differ from lecithins by having a band at 1111 cm-1 and no band at 1176 cm-1. Also, their absorption bands at 1212, 1081, and 1058 cm-I are at slightly lower wavenumbers than the corresponding bands of lecithins. Verma and Wallach 5~ have obtained polarized infrared spectra from multibilayers of lecithin in both the presence and absence of cholesterol, lysolecithin, and phosphatidyl ethanolamine. In the presence of cholesterol (structure given in Fig. 5), the P = O band shifts
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SPECTROSCOPY
131
to a lower position (1230 cm-1), suggesting hydrogen bonding between the phosphatide P-~-O and the cholesterol hydroxyl. A study of the diehroism showed that addition of cholesterol to lecithin multibilayers induees a eonfigurational rearrangement of the polar head groups. Because of hydrogen bonding the cholesterol ring portion extends to C6 from the ester linkages of lecithin, making the proximal apolar acyl chain segments rigid. Infrared spectroscopy has been applied to the analysis of blood lipids. ~t 4. Proteins, Polypeptides, and Related Compounds
Much time and effort have gone into studies of the molecular conformation of proteins, polypeptides, and related maeromolecules. An early review discussing the empirical approach used to study amino acids, polypeptides, and proteins has been given by Sutherland. ~: Sehellman and Sehellman2a and Susi ~3 have discussed developments in the study of conformations of such molecules. Miyazawa and his co-workers 5~-56 used the transisomer of N-methylacetamide and its deuterated form to ascertain the character of the motions associated with the amide I and amide II bands, which occur at about 1650 and 1525 cm-~, respectively, in proteins. In the amide I vibration most of the energy is assoelated with C = O stretching; minor amounts are due to C--N stretching and a bending motion of the N - - H bond. In the amide II vibration, 60 % of the energy is associated with the N - - H bending motion, and the rest is due to the C--N stretching motion. Miyazawa and Blout 57and Miyazawa ~8presented comparisons between observed and calculated frequencies of the amide I and II bands of polypeptides in various conformations-random coil, a-helix, parallel-chain pleated sheet, and antiparallel-chain pleated sheet. (We are using the term "frequency" loosely here according to spectroscopic jargon, but one should remember that we are talking about c m -1, waves/cm.) There was excellent agreement in observed and calculated values. Krimm ~9 presented similar data on the parallel-chain pleatedsheet conformation. Fraser and Suzuki6° studied the dichroism of flkeratin and found three amide I frequencies associated with the antiparallel-ehain pleated sheet. Monosubstituted amides and polypeptides display a strong band at ~3300 em-~ (the amide A band) and a weaker band at ~3100 em-~ (the amide B band). ~8 These bands are associated with the N - - H bond (see Table I). Amide bands III, IV, V, VI, and VII of the peptide group have also been analyzed by Miyazawa. ~8 The amide III band occurs at ~1300 em-~. The amide IV, V, and VI bands are observed at ~620, ~-~700, and ~-~600 em-~, respectively. The amide VII is observed at 206 em-1 (in the Raman effect). Only limited applieatlon has been made of the amide III to VII bands in eonformational studies. The amide V bands are found in the following positions in polypeptides6~: at about 700 em-t for the extended form of poly-3,-methyl-Lglutamate, poly-L-alanine, and sodium polyglutamate; 132
Volume 29, Number 2, 1975
TABLE I. Characteristic Polypeptide Band Frequencies.
Band Amide A Amide B Amide I Amide II Amide III Amide IV Amide V Amide VI Amide -VII
Location (cm-1)• ~3300 ~3100 ~1650 ~1525 ,--4300 ~620 ~700; ~650; ~620 ~600 ~206 (in Raman effect)
These values are approximate. See text and references. at about 650 cm -~ for the disordered form of polyserine and sodium polyglutamate; and at about 620 cm-1 for the a-helical form of poly-~-methyl-L-glutamate, poly~-benzyl-L-glutamate, and poly-L-alanine. The amide V bands of the protein lysozyme are at 690, 650, and 600 cm-~, respectively, for the extended, disordered, and a-helical forms. 62 Examples of the use of far infrared spectra (below 200 e m -1) a r e the studies by Itoh et al., TM ~ who recorded the spectra of the a and ¢~ structural conformations of polyalanines. Many other studies of fibrous proteins, polypeptides, etc., have been done. The reader is referred to Refs. 1, 17, 58, and 61 and references therein. The globular proteins have been studied advantageously in D20 solution, since this solvent affords an excellent infrared window. Timasheff and Susi 6a recorded the spectra of several proteins in D.20, for example, ~-lactoglobulin, myoglobin, and as-casein. Myoglobin in D20 at pD 6.6 has an amide I band at 1650 cm-', characteristic of a-helical structure. Timasheff and Susi's data show that in D20 solution the conformation and the a-helical one produce amide I bands at the same frequencies as in dry films. Randomly folded proteins such as an-casein and denatured /~-lactoglobulin give rise to a sharp band centering at 1643 em-'. Timasheff et al. 66 have reported spectra of the amide I region of many other proteins in D20. Some spectra were also recorded on H20 solutions, mineral oil suspensions, and under other conditions (see also Refs. 67, 68). More recent applications are the study of protein conformation in reconstituted ribosomes 6~ and studies on the conformation of myosin.7° Infrared spectroscopy has been used in examination of the structure of a glycoprotein, bovine submaxillary muein, that had its 0-acetyl groups removed. No absorption in the 1725-cm -~ region was present in the ease of the de-O-acetylated muein, but the native mucin originally had a distinct band near 1725 cm-~. The absence of such a band was a measure of the lack of 0acetyl content of the mucin. 7t Bradbury et al. 72 have studied partial nueleoproteins produced by removal of histone fractions from nucleohistone. A polarized band at 1452 cm-' for one of the nucleoproteins that had been oriented by shearing gave evidenee that the extended polypeptide chain was situated so that its axis lay between the angle of the groove in the DNA double helix and the axis of the DNA helices.
5. Hydrogen-Deuterium Exchange
The hydrogen-deuterium exchange method is a useful tool for studying the tertiary structure of proteins, nucleic acids, and other biopolymers. The method and its applications as performed by means of infrared spectroscopy have been reviewed by several authors ~, n-77 recently. By measuring quantitatively the rate of hydrogen exchange in a given biopolymer under specific conditions, one can obtain a characterization of its conformation (or distribution of conformations) present under these conditions. Lactic dehydrogenase is a good example of an enzyme studied by this method. 7s 6. Nucleic A c i d s and Related Compounds
Much use of infrared spectroscopy has been made in studies of nucleic acids, polynucleotides, nucleotides, pyrimidines, and purines. Tsuboi, 79 in a review, has prepared a table of band frequencies which summarizes much material and has presented spectra of calf thymus DNA and yeast transfer RNA. Shimanouchi8° and Susi ~3have also reviewed this field. An example of the use of polarized spectra was given by Bradbury et al., s' who studied oriented sheets of DNA salts and thereby determined the orientation of the bases relative to the helix axis. Others have done such studies also to determine the geometry of the bases, the sugar, and phosphate groups. 82"84 Falk et al. 85 have studied the infrared spectrum of DNA as a function of relative humidity and concluded that PO~-Na+ groups become hydrated between 0 and 65 % relative humidity, while the bases begin to hydrate above this range. The same workers 86 concluded that DNA films are stable in the B-configuration at relative humidities as low as 75 % and that at still lower humidities a reversible transition occurs to a disordered form in which the bases are no longer stacked one above the other and are no longer perpendicular to the axis of the helix. Tsuboi TM has reviewed work done on double helical RNA, including that by Sato et al. 87 These authors 87 studied the dichroie properties of RNA from rice dwarf virus and used their data to find the geometric parameters governing the orientation of the PO~- group with respect to the molecular axis of the macromolecule. Higuchi et al. s8 have done similar studies on a DNARNA hybrid. They showed absorption bands in their spectra which were assignable to vibrations localized in the base residues. Aqueous solution (D20) spectroscopy has been used in many studies of nucleosides, nucleotides, polynucleotides, and DNA. ~ Integrated absorption intensities for many of these substances have been determined.89 Tautomeric structures have been studied2 °, 91 The interactions of polynucleotide complexes; the analysis of different helical structures in complex mixtures;92, 93 and the interaction of polynucleotides with nucleosides, nucleotides, and purines have been investigated2~ Felsenfeld and Miles 95 and Miles 98 have reviewed these topics. Thomas 97 used infrared spectroscony to determine the percentages of adenine-uracil and guanine-cytosine
base pairs in RNAs containing double helical and single-stranded regions. Klee and Mudd 9s studied the conformations of some adenosine derivatives by several methods, including infrared spectroscopy. These authors also measured the pK ~of compounds by this method. 7. Steroids
Numerous applications of infrared spectroscopy have been made in the field of steroid chemistry. 1Some of the early work has been reviewed by several authors2 ~-10~ Collections of infrared charts of steroids and steroidal sapogenins have been published. 1°2-'°4 A chapter applying conformational analysis to steroids has been published. 1°~ Jones et al. ~°° and Jones and Herling '°7 have given much information on correlations of steroid structures with their spectra. The reader is referred to Ref. 1 for a review of the work of many authors concerning the identification of carbonyl and hydroxyl groups, as well as unsaturation in various parts of the steroid nucleus. Hydrogen bonding, the measurement of integrated absorption intensities, and the application of infrared to metabolic studies are also discussed. A graphical method for predicting the characteristics of a steroid spectrum has been developed. 1°8 8. P o r p h y r i n s and Related Compounds
Schwartz et al. 1°9 have published tables of characteristic group frequencies of porphyrins and low frequency assignments for porphin and tetraphenylporphin. Marks ~1°has discussed the physical properties of tetrapyrroles and has given infrared data for porphyrin and heine esters. Boucher and Katz TM have studied the infrared spectra of complexes of divalent metals with protoporphyrin IX derivatives. Protoporphyrin IX is shown in Fig. 6. Caughey el al. m made vibrational assignments for most of the peripheral ring substituents of a series of substituted deuteroporphyrins IX and for bands of the porphyrin ring as a whole. Alben et al. m have reviewed their work with porphyrins. They have recorded infrared spectra of porphin, etioporphyrin, tetra-(normal-propyl) porphyrin, and tetraphenylporphyrin; m e s o - t e t r a p h e n y l p o r p h i n and its nickel (II) and copper (II) complexes, m e s o - s u b s t i t u t e d porphin derivatives and their iron (III) mu-oxo com-
CH=CH2[ ~H3 ~C\ H ~C~ C
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~H2
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FIG. 6. Structure of protoporphyrin IX. APPLIED SPECTROSCOPY
133
plexes; deuteroporphyrin derivatives and their nickel(II) complexes; and 2,4-diacetyldeuteroporphyrin IX dimethyl ester (1-2 H) and its metal complexes. In addition to finding information concerning the assignment of various vibrational modes, these authors have studied the molecular interactions between one part of the molecule and another, e.g., between the pyrrole fl-carbon substituents and the metal groups in the center of the porphyrin, and between the coordinated metals and the peripheral parts of the porphyrin molecule. Caughey and his co-workers"~, ~s have studied the binding of carbon monoxide to heroes, hemoglobins, and myoglobins. Fager and Albert ~ have studied the structure of the carbon monoxide-binding site of hemo~ cyanins. A model structure which they proposed for the hemocyanin carbonyl complex includes a trigonal oxygen of CO coordinated to one copper atom, with the second copper atom of the binding site coordinated only to protein. This study used Fourier transform infrared spectroscopy on a Digilab FTS~14 instrument. Berzofsky et al. ~ have also used the FTS-14 to investi~ gate carboxysulfmyoglobin and the relation between electron withdrawal from the iron atom and ligand binding. Katz et al. ~s and Marks ~° have reviewed the infrared spectroscopy of chlorophylls and related compounds. More details concerning this subject and porphyrins in general are given in Ref. 1. 9. E n z y m o l o g y
Infrared spectroscopy has been used advantageously in studies of enzymatic reactions and of conformations of enzyme structure? Examples of studies of the mechanisms of enzymatic action are work on carbonic anhydrase, "~ arylsulfatase,~° ~-methylaspartase, TM and rhodanese?~ Examples of enzymes on which conformational work has been done by the infrared method of hydrogen-deuterium exchange are the following: ribonuclease, ~ glyceraldehyde 3-phosphate dehydrogchase, ~ trypsin, 1~ a-chymotrypsin,~ lysozyme,~ alcohol dehydrogenase,~s and k-glutamate dehydrogenase?~ Infrared spectroscopy has also been applied to studies of enzyme inhibition, enzyme kinetics, and the identification of microorganisms by their enzymic action2 Chapman et al. ~° differentiated between bacterial fl-lactamase and amidase activities by virtue of infrared spectral changes of antibiotic substrates exposed to them. 10. A p p l i c a t i o n s i n M e d i c i n e and Related Fields
Infrared spectroscopy has been used in many ways in clinical laboratories? The infrared analysis of serum and other fluids from healthy individuals and patients with various diseases has been an aid in the I diagnosis of those diseases, lal Normal and diseased skin surfaces have been examined by the internal reflection method. ~ Urines of normal and of diabetic persons have been examined?~ Serum lipoproteins have been quantitated.~l. ~ The qualitative analysis of human biliary calculi (gallstones) has been carried out. ~ Renal calculi 134
Volume 29, Number 2, 197S
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Fro. 7. Infrared spectrum of aqueous citric acid, 10% (w/v).1~ (kidney stones) have also been analyzed. ~36 Infrared techniques have been used for detecting toxic gases and volatile organic compounds in expired breath, blood, urine, and tissues. ~37 A method has been described for monitoring the composition of gaseous mixtures of volatile anesthetics used in anesthesiology,la8 Mills et al. ~89 have discussed the use of infrared spectroscopy to differentiate brain tissues of patients with Huntington's chorea from those of patients with nonhereditary degenerative disease. Gimeno Ortega et al. 1~° have used an infrared analyzer for the determination of the carbon dioxide content of blood. 11. Other Biochemical Topics
This is a very broad field. Many of the subjects in this category have been reviewed by Parker? Among them are aqueous-solution infrared spectroscopy, a field of investigation in which much work has been done with both H20 and D20; metal chelates and complexes; biological membranes; tissues, normal and cancerous; red blood cells of various animals; bone and collagen; teeth and bone minerals; vitamins, hormones, and coenzymes; quinones; and insect sex attractants. Gangloff et al. 14~have applied data obtained by Parker t42to determine tricarboxylic acid cycle patterns in liver and muscle tissue in hypothyroid and hyperthyroid animals. Fig. 7 shows the spectrum of 10 % aqueous citric acid, a tricarboxylie acid. ~2 Recently, Fourier transform spectroscopy has been applied to a study of aqueous solutions, e.g., sodium citrate and other compounds in a variety of concentrations. 143 II. R A M A N S P E C T R O S C O P Y A. Sampling M e t h o d s and S o m e General Considerations
In some respects it is more difficult to handle samples for Raman spectroscopy than for infrared. In the former the purity and homogeneity of samples must often be better controlled than in the latter. On the other hand, Raman spectroscopic sample cells, usually made of glass, lend themselves more readily to the use of aqueous solutions. Powders can be sampled by direct
scattering off their surface, or they may be packed in melting point capillaries and illuminated either horizontally or vertically. Small crystals can be contained in capillary tubes for sampling. If a crystal is large enough to hold, one can sample it by holding it directly in the laser beam. (Most of the work we shall refer to will be laser-Raman spectroscopy.) For solid materials one does not have to grind the sample and consequently destroy its integrity. Sample thickness is not a problem since Raman spectroscopy is essentially akin to an emission process rather than an absorption phenomenon. A problem with sampling in Raman spectroscopy is the fluorescence one may produce when exposing a sample to laser excitation. For example, the disadvantage of the argon ion laser is in the greater probability of fluorescence and sample decomposition from shorter wavelength excitation. However, using a He-Ne laser source (red excitation), Lord and Yu TM found no serious fluorescence problems in the examination of the enzyme lysozyme and its constituent amino acids. Sometimes the fluorescence seen in Raman spectra of biochemicM substances is not caused by the materials themselves but rather by organic impurities. These impurities are often "removed" by long exposure to the source. Commercial samples are sometimes rendered spectroscopically acceptable by treatment with activated charcoal. Filtering solutions through fritted glass discs or centrifuging prior to filling sample cells minimizes Tyndall scattering, which is caused by dust, suspended material, colloids, bubbles, or some other undissolved material with particle size about the same or greater than the excitation wavelength. Absorption of the excitation wavelength by the sample and fluorescence caused by this wavelength are to be avoided, since these processes make it difficult to obtain adequate Raman spectra. Also, conditions should be optimized for obtaining as strong Raman scattering as possible, since the Raman effect is basically a weak phenomenon. Bailey et al. '4~ and Hawes et al. ~4~ have described several spatial arrangements for excitation of relatively small amounts of sample, and these arrangements produce efficient scattering. Raman speetrophotometers are essentially "single beam" instruments, and therefore, unlike the situations with infrared double beam instruments, they cannot be used for differential spectroscopy to compensate for Raman scattering of solvents and such things as impurities. To compensate for a solvent, one has to resort to the method used for the early single beam infrared instruments: one substracts the spectrum of the solvent from the spectrum of the solution, each spectrum determined separately. Jones et al. 147 discussed the use of an internal standard for doing quantitative work. Lord and Thomas ~48normalized all Raman intensities to a single Raman line (459 cm-~ line of carbon tetrachloride) for quantitative examination of cytidine and HgC12 solutions. Several authors have discussed the quantitation of Raman intensities?47, m-1~2Raman band intensities are essentially
linear with concentration and not logarithmic as in infrared (or other) absorption spectroscopy. The intensities are also linear with respect to laser power and volume of the sample excited. Several authors have discussed general experimental techniques for Raman spectroscopy. 147, 153-~55
B. Polypeptides, Proteins, Enzymes, and Related Substances Although much work with aqueous systems has been done by infrared spectroscopy (see Ref. 1 and the citations therein), the Raman effect has the advantage that there is little interference by the solvent. In the l~aman spectrum of water there is only one moderately intense band in the region 3200 to 3600 cm-1, and other extremely weak water bands occur near 1640, 800, 450, and 175 cm-k Thus, the biochemist can study systems that may be difficult or perhaps impossible by infrared methods. However, the large volume of reports over the years on the excellent use of water and D20 for infrared work should be kept in mind and the methods not dismissed lightly. Lord ~6 has reviewed Raman spectroscopic methods and has discussed results obtained in his laboratory and others on amino acids, synthetic polypeptides, proteins of low molecular weight, nucleic acids, and polynucleotides. References to work of the 1930's and the more recent technique of laser-Raman spectroscopy are given in this paper. Several authors have reported high intensity background in the Raman spectra of proteins and polypeptides. The origin of this background is controversial, since it has been tentatively attributed to fluorescent impurities, ~ to lI~aman scattering, 1~8,~9 and to the lack of optical homogeneity of the sample?44 Careri et al26° have discussed the broad, intense scattering background of aqueous protein solutions and have measured the decrease of this background as a function of the irradiation power density. They have also pointed out some implications of the background recovery in the dark. They showed that the spectrum of a 2 % bovine serum albumin solution, irradiated for 10 h, had its scattering background practically eliminated, but that after 10 h of storage in the dark, the spectrum recovered the spectral properties of a control nonirradiated solution. Other evidence, suggesting that the physical process causing the background cannot be identified as a simple Raman or fluorescent process, has been stated in another report26~ Koenig~62 has reviewed the Raman spectroscopy of certain biological molecules, namely amino and imino acids (see Fig. 8); oligopeptides and polypeptides (including discussions of the a-helical and ~-eonformations, other helical forms, and random coil configuration); and proteins, including some enzymes. Raman spectra of polyglycines, poly-L-alanine,~, ,64 poly_L_proline,l~7 and poly-L-lysine~65 have been obtained both in the solid state and in solution. Koenig et al. ~57, ,63. ,~4. ,67-~6s have found that the amide III line in polypeptides with the a-helix structure occurs at frequencies different from those with the G-pleated sheet or random coil APPLIED S P E C T R O S C O P Y
135
H2
jC
H2C \ H CH3CHCOOH
C--COOH I ] H2C~N/HI H
o
b
Fro. 8. a, alanine, an amino acid; b, proline, an imino acid.
structures. Among the substances studied were polyglycines, poly-L-proline, and poly-L-alanine. Lord and Yu TM have given a table of Raman spectral data for some simple peptides in water and D20 at a variety of pH and pD values. The compounds studied were glycylglycine, triglycine, L-alanylglycine, and glycyl-L-alanine. These authors have also prepared a table of 20 amino acid spectra at various pH values in the range of 200 to 2000 cm -~. They obtained laserexcited (He-Ne) spectra of the enzyme lysozyme in aqueous solution and partially interpreted them by using spectra of the constituent amino acids. These authors stated that Raman spectroscopy should be useful in providing direct evidence concerning the presence and number of disulfide cross links in proteins, and may also be useful in studying the local conformation of the C - - S - - S - - C group. Phenylalanine, tryptophan, and tyrosine aromatic groups produced very intense and sharp Raman lines, which were not sensitive to changes in conformation or state of aggregation. The peptide group (CONH) produced two characteristic lines near 1660 cm-~ (amide I) and 1260 cm-t (amide III). These lines can be used for studying eonformational changes resulting from denaturation. The region 800 to 1150 cm -~ showed lines produced by C--C and C - - N stretching vibrations, and these lines were expected to be dependent on conformation. Information about the backbone conformation of proteins and polypeptides may be obtained from the amide I (1630 to 1700 cm-~) and amide III (1220 to 1300 cm-1) regions of Raman spectra, m, 16~.169.~70 Yu and Liu 169 studied the Raman spectra of glucagon in various conformations. They concluded that the ahelical, random-coiled (hydrogen-bonded), and antiparallel 9-structure of a protein should have the amide I frequencies at 1660, 1665, and 1672 cm -~, respectively, and that the corresponding amide III vibrations should be 1266, 1248, and 1232 cm-1. Yu et al. 171 observed a strong single line at 1672 cm -~ in both solid and solution and a resolved peak at 1238 cm -~ in solid cobramine B, a small basic protein from cobra venom, and at 1235 cIn-1 in solution. They concluded that this substance contains a large fraction of antiparallel p-structure. Other resolved peaks at 1251 in the solid and 1254 in solution and 1270 cm-1 in both phases indicated that random coil and a-helix were also present. These authors also found that all three tyrosines in this protein appear to be "buried" in the interior of the molecule and are probably involved in interactions which are similar to those of the three "buried" tyrosines in RNase A when it is dissolved in water. The 136
Volume 29, Number 2, 1975
S--S and C--S stretching vibrations of three disulfide linkages in cobramine B appeared, respectively, at 510 and 675 em-L Tobin 172 used laser excitation (He-Ne 632.8 nm and Ar 514.5 nm) and obtained Raman spectra of crystalline a-chymotrypsin, pepsin, and lysozyme. He found 21 Raman lines for lysozyme and gave provisional assignments as follows: 1430 cm-1, CH; 1512, amide NH; 1625, C-~-C; 1667, amide C-~O; 2870, 2926, and 2964, aliphatic CH; 3053, aromatic CH; and 3300 cm -I, NH. Lord and Yu m recorded Raman spectra of native ribonuclease, a-chymotrypsin, and poly-L-glutamic acid in aqueous solutions. The strong Raman lines of the aromatic side chains of the amino acid residues were not affected by conformations of the proteins. The disulfide link showed a bond-stretching vibration in the spectrum of ribonuclease at 516 cm -~, but at 509 cm-' in lysozyme. The intensity ratio of the C--S to S--S lines was an order of magnitude larger than in lysozyme. These observations suggested that the conformations of the C - - S - - S - - C cross-links in the two enzymes are significantly different. When these workers observed the spectrum of poly-L-glutamic acid at pH 10 as a possible model for denaturation effects, they found that comparison of the spectra of the native enzymes with that of the polypeptide showed that the latter displayed broadening and overlapping of peaks in the amide I and amide III regions which were sharper and resolved into several components in the spectra of the proteins. Spectra of the denatured proteins had not been obtained at that time. Brown el at. 17~ obtained low frequency Raman spectra from a variety of a-chymotrypsin samples. They found a peak at about 29 cm-1 for all the samples except the one that had been denatured by sodium dodecyl sulfate. These workers attributed such low frequency bands to vibrations that involve all or very large portions of the protein molecule. Pepsin shows a strong band at about 32 cm-', which disappears upon heat denaturation. Thus, these frequencies are dependent upon the conformation of the protein molecule. Lord and Mendelsohn 17~ found that 6 M LiBr, whose ions in solution affect the Raman spectrum of water relatively little, can be used to denature lysozyme without obscuring the enzyme's Ilaman spectrum. These authors recorded spectra of lysozyme in the range 1200 to 1500 cm -1 at several concentrations of LiBr. In the range from 4 to 6 M the shift of the amide III band center of gravity was from above 1260 cm-~ to about 1245 cm-'. This shift was interpreted as a removal of the ordered structure in the protein backbone, with only random coil structure remaining. Denaturation also caused the S--S frequency at 509 cm- ' to broaden and weaken, a fact interpreted to mean that the dihedral angles around the four S--S bonds varied considerably more than in native lysozyme. Brunner and Sussner 176 obtained Raman spectra of lysozyme in aqueous solutions. Several spectral lines due to vibrations of aromatic residues or the protein backbone changed in frequency and/or intensity when the enzyme was thermally denatured. The line at 504
em-1 (owing to the disulfide stretching vibration of the cystine residues) essentially disappeared above 76°C, probably signifying the breakage of all disulfide linkages in the denatured state. Yu et al. m presented spectra of a model protein, ribonuclease A, in the powder form and in aqueous solution. They compared these spectra quantitatively and interpreted the spectral differences in terms of main chain and side chain conformational changes. A tentative conclusion was that the intensity of Raman bands of the ring vibrations of tyrosine is sensitive to changes in conformation. Also, the geometry of the disulfide linkages is more uniform in solution than in the solid state, and the C--S--S--C bond angles are smaller in solution. Yu et al. '78 recorded spectra of native and denatured insulin in the solid state and in aqueous solution. Denaturation caused marked spectral changes in the following regions: amide I and amide III; S--S and C--S stretching; skeletal bending; and skeletal stretching. Extensive conformational changes occurred in the conversion of native to denatured fibrous insulin. Proinsulin and insulin spectra showed 11 conformational-depcndent lines in good agreement with one another, suggesting that the insulin moiety of proinsulin exists in a conformation very nearly the same as that of insulin itself. Graphical subtraction of the two spectra yielded a peak at 1663 cm -1 with a shoulder near 1685 cm-1. The first line is indicative of a-helical structure and the second of random coil form in the C-peptide (the Cpeptide is composed of 30 amino acid residues and is removed as a single chain when proinsulin is converted to insulin). Yu et al. ~79 presented a Raman spectrum of rhombohedral zinc-insulin crystals. The S--S bond-stretching vibrations of the three disulfide bonds appeared at 517 and 505 cm -1. Two C--S stretching frequencies appeared at 668 and 680 cm-1 and were assigned to the C--S stretching vibrations of the C--S--S--C groups. Based on these observations and other work on model compounds, the 505 and 680 cm-1 lines were assigned tentatively to the intrachain A6-11 disulfide link and the other pair at 517 and 668 cm-1 were assigned to the interchain links, BT-A7 and B19-A20. The amide III region consisted of three resolved peaks at 1239, 1270, and 1288 cm-~, indicating the presence of three distinct structural components in the polypeptide chains of the insulin crystals. The question of whether the structure of a globular protein is the same in crystals as in solution has been examined. Yu and JotS° studied the amide I and amide I I I regions of the spectra of lysozyme chloride crystals (100 % relative humidity) and of lysozyme in solution at pH 4.50. They found good agreement between these spectra, the data indicating that the main-chain conformation is the same in both phases. Small spectral differences were found near 464, 622, 644, 934, 960, 978, 1032, 1129, and 1196 era-!, some of which were interpreted in terms of side chain eonformational changes. Also, lyophilization caused appreciable eonformational changes in both main and side chains. Yu and Jo 18~ presented the Raman spectra of ribo-
nuclease A in several states: single crystal, crystalline powder, lyophilized powder, and aqueous solution of pH 5.71 (see Fig. 9). The spectra of the single crystal and of the crystalline powder in the 500 to 700 cm-~ region (where the S--S and C--S stretching and the tyrosyl ring vibrations occur) were somewhat similar to that of lyophilized powder but different from that of the aqueous solution. These workers interpreted the spectral differences between crystal and solution phases to be the result of changes in the geometry of the disulfide linages and the local environment of the so-called buried tyrosines upon crystallization. Data for the amide III region indicated that the backbone conformation of RNase A is the same in the crystal and in solution. Raman spectra of carboxypeptidase A in crystal line form and in solution showed a small difference in the line shape of the amide III region, which may be a reflection of subtle backbone conformational changes. Bellocq el al. ~82 obtained Raman spectra of aqueous bovine serum albumin and ~-lactoglobu]in in the range 500 to 1600 cm-~. They found about 30 lines for each protein, half of which were assigned to functional groups of amino acid residues. ¢~-Lactoglobulin was predominantly in a random coil form. The albumin had a higher proportion of ordered structure. Infrared and lI~aman spectra were obtained by Fcrmandjian et al. 's3 for angiotensinamide II (an octapeptide), some constituent peptides, and an analog. These workers determined amide vibrations of the solid hormone in the 500 to 1700 cm-~ range. Angiotensinamide II had a preferential antiparallel/F-conformation in both the solid state and concentrated aqueous solutions. Raman spectroscopy has been used to study different conformations of the antibiotic valinomycin.TM This compound (which is composed of 3 moles of L-valine, 3 moles of D-valine, 3 moles of L-lactic acid, and 3 moles of D-c~-hydroxyisovalerie acid, linked alternately by ester and amide bonds to form a 36-membered ring) has ionselective carrier properties in biological and artificial membranes. The spectrum of crystals grown from both n-octane and acetone showed splitting of the ester and amide earbonyl stretching vibrations, observations which support the contention that some of the ester earbonyl groups are intramoleeularly hydrogen-bonded in valinomyein. C. Nucleosides, Nucleotides, Nucleic Acids, and Related Substances Lord and Thomas, 185using 435.8 nm mercury arc excitation, recorded the Raman spectra of the commonly occurring purine and pyrimidine derivatives of ribonucleic acid. They examined the free bases, nucleosidcs, 5t-mononucleotides, and related alkyl derivatives in aqueous solutions over wide pH and pD ranges as well as in the solid state. The region of great usefulness for these investigations was that from 1800 to 1500 cm-1, which includes characteristic frequencies of the double bonds C=O, C-~N, and C = C . Other frequencies below 1500 cm-1 were also characterized, e.g., ring modes, vibrations of the phosphate and sugar residues, and APPLIED SPECTROSCOPY
137
crystalline state. The hydrogen bond-stretching frequencies for these crystals were found in the range 125 to 70 cm -I and showed up only feebly in the Raman effect, whereas the out-of-plane molecular librations were quite intense. These librations and the translational lattice modes had frequencies below 100 cm-L Intramolecular out-of-plane ring deformation vibrations also appeared in the far infrared spectra in the range 220 to 135 cm-L Raman spectra of aqueous solutions of inosine (Fig. 10) and its methylated analogs have been obtained, m9 A broad line near 1680 cm -~ was due mainly to the carbonyl stretching vibration and it identified the keto structure. ~9° Also, the absence of a line at 736 cm -1 in the spectrum of inosine allowed the conclusion that the tautomeric equilibrium lies very strongly in the direction of the keto form, since an enol analog (6-methoxypurine riboside) displayed an intense band at 736 cm-L Inosine, its 5'-monophosphate, and 1-methylinosine have also been studied b y Raman spectroscopy TM with respect to associative interactions.
deformation modes. This was a very detailed investigation, with m a n y spectra and tables of data reported. Lord and Thomas 186 studied Raman spectra of aqueous solutions and crystalline derivatives of uracil, adenine, guanine, and cytosine. T h e y discussed tautomeric effects, structural effects of pH, nucleotide derivatives, and metal-to-nucleotide binding. Arie et al. ~87 studied the spectra of uracil, guanine, and cytosine as powders and the two pyrimidines in acidic, neutral, and alkaline aqueous solutions. Lord and Thomas, ~48using a mercury arc, studied the Raman spectra of complementary base pairs of purine and pyrimidine nucleoside and nucleotide derivatives in aqueous solutions. T h e y found no evidence for specific base-pairing interactions but were able to detect interactions between nucleosides and heavy metal ions, e.g., the formation of a cytidine-HgC12 complex. Harada and Lord m8 determined the low frequency infrared and Raman spectra, 30 to 230 cm -~, of 1methyluracil, 1-methylthymine, 9-methyladenine, and he 1:1 complex of the last two compounds in the
(a)
Solution pH 5.71
RNase
A Amld. I
$-$
~7 C-${cv,1
Jl ,4 l ~"
Amlde III
':2?x "
..
PhelV I C:S (M,')
123~26S
Tyr
~
903
fl.,:?/
° ' i ~ +,+] ~
l/
J
t
J >l--
m (b)
Lyophilized Powder
.
,~,
P
J ,
[ 600
,
I 800
,
I 1000
,
l 1200
,
] 1400
y ,
I , 1600 ctw 1
FIG. 9. The striking effect of lyophilization on the conformation of RNase A. a, Raman spectrum of RNase A in aqueous solution: concentration, 150mg/ml; pH 5.71; spectral slitwidth (Aa), 4 cm-1; sensitivity (s), 1000 counts/see full scale; rate of scan (,~), 10 cm-1/min; standard deviation (Sd), 0.7%; laser power (p) at the sample, 100 roW. b, Raman spectrum of RNase A lyophilized p o w d e r at 0% relative humidity: Ao-, 4 cm-1; s, 5000 cps; "~, 10 cm-1/min; Sd, 0.7%; p, 153 roW. Reprinted with permission from Ref. 181. Copyright by the American Chemical Society. 138
Volume
29, N u m b e r
2, 1975
0
HO(~H2
I
OH OH Fm. 10. Structure of inosine. Rimai et al. 192 obtained Raman spectra for adenosine tri-, di-, and monophosphate in aqueous solution at pH range 0.5 to 13.5 and between 550 and 1700 cm-L The spectra were pH-dependent. Assignments were made of vibrations of the phosphate complex. ADP was distinguishable from ATP by the Raman technique. Lines at about 960 and 1100 cm-~ were good indications of the degree of ionization of the terminal phosphate group. Heyde and Rimai ~93 measured the frequency of the 1125 cm-~ line of the phosphate group of ATP as a function of pH in aqueous solutions of ATP alone and of ATP complexes with Na +, Mg 2+, and Ca 2+. From these data they determined the pK, of HATP 8- and the stability constants of ATP-metal complexes, where the metals were those mentioned above. The 1125 cm-~ line is associated with the P = O bond stretching motion and corresponds to a symmetric vibration. It was used here as a conformational indicator. Lord and Thomas ~85have provided a comprehensive catalog of the Raman spectra of the monomeric units of RNA. Knowledge concerning these units allows analysis of the Raman spectra of synthetic polyribonucleotides and their conformations in aqueous solution. The same authors 18~ examined polyriboadenylic acid poly(rA). Broader studies have been done by Fanconi et al. TM and Small and Peticolas ~95 for poly(rA) and for poly(rI), poly(rC), poly(rG) and poly(rU). The latter investigators have shown that the Raman lines at 1508, 1303, and 725 cm-1 in poly(rA), characteristic of the adenine moiety,1s5 have a marked temperature dependence, which is considered to be sensitive to conformational changes. These workers have related intensity changes of certain Raman lines to hypochromic spectral effects. Rice et al. ~96 have observed that Raman spectra of polyriboguanylic acid, poly(rG), obtained from neutral, aqueous solutions containing excess Na + counterions, were qualitatively different from spectra of salt-free solutions. A twofold molar excess of Na + (per phosphate group) produced a spectrum of poly(rG) characteristic of an ordered helical structure having H-bonding between guanine residues. The absence of added sMt yielded a spectrum of poly(rG) suggesting either a tautomeric or zwitterionic modification of the ordinary (keto-amino) ring structure. 91 Lafieur et al. 19~ have reported spectra of the double helical complexes poly (A).poly(U) and poly(G).poly(C) and have discussed them in relation to their use as quantitative reference spectra for determining the dependence of the Raman scattering of RNA on secondary structure.
Koenig, 162in a review referred to earlier, has discussed DNA, RNA, synthetic polyribonucleotides and polydeoxyribonucleotides, and purine and pyrimidine bases. Morikawa et al. 19s have shown that Raman and infrared spectra of aqueous poly(rA-rU).poly(rA-rU), the double helical complex containing strands of alternating riboadenylate and ribouridylate residues, display significant differences from one another and from corresponding spectra of poly(rA).poly(rU), the double helical complex of riboadenylate and ribouridylate homopolymers. The differences observed between vibrational spectra of poly(rA-rU).poly(rA-rU) and poly(rA). poly(rU) are not caused by different base-pairing patterns but may be due to differences in vibrational coupling between vertically stacked bases. Spectral and other data indicated that Raman and infrared frequencies of RNA in the region 1750 to 1550 cm-1 should be dependent on the sequence of bases. Hirano 199 presented preliminary Raman spectra of DNA in aqueous solution. He recorded several broad lines, but fluorescence may have accounted for these. Erfurth e t a / . 2°° prepared fibers of DNA in the A, B, and C forms and compared Raman spectra of these fibers with those of calf thymus DNA and yeast transfer RNA in dilute solution. They showed that the phosphate vibrations in the region 750 to 850 cm-1 are very sensitive to the specific conformation of the phosphate group in the backbone chain and are virtually independent of all other factors. The Raman method introduces an easy technique for the determination of the specific conformation of the sugar-phosphate backbone chain of nucleic acids. This method is applicable to single chain structures, oligomers, and nucleic acids in viruses and nucleic acid-protein complexes. The method of handling samples and instrumentation for Raman spectroscopy of RNAs have been described by Thomas e t a / . 2°1-2°3 Thomas et al. ~°~ have found that tRNA fM~t,tRNA TM, and tRNA~ h~from E s c h e r i c h i a coli have similar conformations in aqueous solution. The spectra of the first two tRNAs showed that their specific base-paired and base-stacked secondary structures were consistent with cloverleaf models proposed by other workers. Additional tRNAs were examined in another study, 2°4 which showed that the presence of excess Mg 2+ caused gross conformational changes in tRNAWL The presence of dihydrouridine (Fig. 11) can be detected in tRNA by means of a Raman spectrum. Hartman et al. 2°5 studied the R17 virus and its RNA. The spectrum of the virus contained many lines assignable to scattering by vibrations of the nucleotide o
HN'J'[~ H2
OH
OH
Fza. 11. Structure of dihydrouridine. APPLIED SPECTROSCOPY
139
residues of RNA and the amino acid residues of protein subunits. These authors compared the Raman lines from specific nucleotide vibrations in the phage with their counterparts in the spectrum of protein-free RNA. Many similarities were suggested between RNA structure in the phage and protein-free forms. The average configuration of guanine residues in the phage was apparently very different from that of protein-free RNA. This finding suggested that guanine plays an important role in RNA-protein interactions. The experimental results of this work indicate that the Raman spectrum of a native virus can display many characteristic vibrations of both its protein and nucleic acid moieties and that Raman spectroscopy is a useful method for the study of virus structure in an aqueous medium. Applications of the technique to ribosomes and chromosomes are also possible. Thomas TM has obtained Raman spectra of ribosomal RNA from E. coli in the range 2000 to 200 cm-~ in aqueous solutions. Raman lines of the phosphodiester group (Fig. 12) and of paired and unpaired purine and pyrimidine bases were readily detected, and their assignments were made. Characteristic ring vibrations allowed each of the four heterocyclie bases to be distinguished from one another in a single spectrum. The symmetric stretching vibration of the O--P--O group at 814 cm-~ was particularly sensitive to ionic strength, probably reflecting changes in the conformation of phosphate groups. Thomas et al. ~°~ found that intensities of Raman scattering from particular vibrations of nucleotide residues in 16 S and 23 S ribosomM RNA are sensitive to changes of RNA conformation caused by temperature changes. These intensity effects are most likely due to a specific RNA tertiary structure. Thomas et al. ~°~ compared Raman spectra of yeast tRNAs in charged and uncharged states to reveal differences in base stacking. Aminoacylation of unfractionated tRNA reduced the amount of stacking of both adenine and pyrimidine residues. However, in Phe-tRNA T M only the adenine residues appeared to be significantly less stacked after aminoacylation. The over-all degree of order in the backbone of yeast tRNAs was little affected by these changes in base-stacked secondary structure. D. Carbohydrates Koenig'62 discussed the Raman spectra of carbohydrates ill aqueous solution as well as in the solid state. Spectra of glucose, maltose, cellobiose, dextran, amylose, amylopectin, glycogen, and cellulose were given. Also given were assignments for the various vibrations observed, including a- or ~-configuration at the C-1 anomeric carbon atom (see Fig. 13). H
0 H
i -
I
I[
2
0
FIG. 12. Structure of the phosphodiester group linking two sugar molecules.The 3'-carbon of one pentose is linked through the phosphodiesterlinkage to the 5t-carbon of the next pentose, 140
Volume 29, Number 2, 1975
C-I anomeric carbon atom / of sugar residue HOCH2
X
~7
/ \oH
FiG. 13. Structure of the a- and B-configurations at the C-1 anomeric carbon atom. The configuration as given is the a-one. Reversal of positions of the H and OH on C-1 gives the G-configuration. E. Steroids
Schrader and Steigner 2°7pointed out that infrared and Raman spectroscopy yield complementary information about steroid molecules. Infrared gives the vibrations of the polar substituents; Raman gives mainly nonpolar skeletal vibrations. The Raman spectrum shows very clearly nonpolar multiple bonds and the arrangement of conjugated systems. Also, the linkage of the A and B rings can be predicted (see Fig. 5 for steroid structure). The same authors 2°8 gave simple rules for applying Raman spectroscopy to the elucidation of steroid structurM characteristics. The rules derived for steroids are also applicable to other classes of molecules. F. Studies of Membranes, Lipids, and Related Substances
Wallach2°9 reviewed the subject of infrared and laserRaman spectroscopy as applied to the analysis of membranes. He discussed soluble polypeptides and proteins generally, membrane proteins, conformational changes, and lipids as studied by both techniques. Mendelsohn21° studied egg lecithin and egg lecithincholesterol mixtures (in a 1:1 molar ratio) by laserRaman spectroscopy. The substances make a simple membrane model system. From the positions and intensities of the Raman lines near 1100 era-' he deduced that the hydrocarbon chains for lecithin were in a liquid conformation. When he added cholesterol, the formation of certain gauche isomers was apparently inhibited and this addition caused a marked increase in chain rigidity. The results supported a theory recently proposed TM for the mechanism of cholesterol-phospholipid interaction. The cholesterol was proposed to insert itself nearly parallel to the hydrocarbon chains so that its ring region is tightly packed with the upper half of the chain (i.e., the half nearest the head groups). The tail region of cholesterol interacts with the lower region of the hydrocarbon, which can then assume gauche conformations, as it is not tightly packed. Lippert and Peticolas2'2 gave Raman spectral assignments of long chain fatty acids which can be used to determine all-trans chain length and the position and configuration of double bonds in homogeneous fatty acid samples. Also, the carbon-carbon stretching region of phospholipids and fatty acids having double bonds shows two intense bands (between 1000 and 1130 em-1) which these authors assigned, respectively, to the alltrans-methylene chain between the acid moiety and the double bond (bound chain) and the methylene chain extending from the double bond to the terminal methyl
group (free chain). They used these bands to study the side chain melting transition of L-a-dioleoyl lecithin suspensions. Both free and bound chains transformed from the all-trans to a random configuration at the same temperature, but the Raman intensities indicated that the interior of the multilayer of dipalmitoyl lecithin is more mobile than the region closer to the polar surface. These authors had previously illustrated the usefulness of laser-Raman spectroscopy for obtaining the conformation of the hydrocarbon chains in dipalmitoyl lecithin multilayers.213 Brown et al. TM investigated the spectral region from 30 to 3300 cm-1 for concentrated samples of dipalmitoyl phosphatidyl choline (Fig. 14) and dipalmitoyl phosphatidyl ethanolamine in aqueous solutions over a range of 90 to 19°C. Frequency shifts occurred in the P O c symmetric stretch band which suggest a change in exposure of the P02 group to the water upon melting. The frequency of the translational hydrocarbon mode around 150 cm-~ appeared to indicate the degree to which the hydrocarbon chain is extended. The methyl and methylene stretch bands at 2890 and 2850 cm-~ demonstrated hydrocarbon chain melting. The 720 cm-~ band, assigned earlier to the symmetric O--P--O diester stretch, 213 appeared to be caused instead by the symmetric C--N stretch of choline. Bulkin2~5recorded the Raman spectrum of human red blood cell membranes in the 1000 to 1500 cm-t region. He found bands at 1110, 1340, 1420, and 1445 cm-~, all attributable to the hydrocarbon chains of the fatty acids, and possibly partially to CH2 groups of cholesterol. The presence of the 1110 cm-1 band indicated that the fatty acid chains are fluid in erythrocyte membranes. Sample volumes of less than 1 gl were used. Conformation-dependent features in the Raman spectra of some simple lipids have been examined by Larsson,2~6 who studied a series of n-paraffins, n-fatty acids, n-alcohols, amides, wax esters, and glycerides in different states. This author had reported earlier~7 that the appearance of the C - - H stretching vibrations in the Raman spectra of lipids depend upon the hydrocarbon chain arrangement, and he stated 2t6 that hydrocarbon chain mesophases show vibration characteristics similar to the liquid state of n-paraffins. Bulkin and Krishnan 21s had shown that cholesteric liquid crystals show differences from the isotropic liquid phase in the 2800 to 3100 cm-~ region. The most sensitive part of Larsson's2~6 spectra for studying the conformation in hydrophobic regions of lipid systems was produced by the C - - H stretching vibrations. Different close-packing arrangements of the hydrocarbon chains could thus be identified in the solid state, and the successive increase in the degree of disorder at transitions into liquid-crystalline CH2OCORI I R2COOCH 0 I II
CH2OI~OCH2CH2N(CH313 O"
FiG. 14. Structure of dipalmitoyl phosphatidyl choline. I~~ and lIt,2 are palraitic acid residues.
and mieellar phases could be seen from changes in the spectra. Well resolved spectral bands were observed for the lipids even when the phases contained more than 90% (w/w) of water, thereby showing that direct analyses of biological systems are possible. Larsson compared Raman spectra of normal human skin and psoriatic skin in the 2900 cm-1 region. The C - - H stretching vibration region for normal skin displayed two peaks at about 2850 and 2890 em-t, respectively, corresponding to lipids with liquid hydrocarbon chains. Psoriatic skin showed one band only at about 2860 em-1. These differences were reproducible on different patients and controls, showing that there are differences in hydrocarbon chain structure or conformation of the lipids in stratum eorneum between normal and psoriatic skin. G. Calcified Tissues
Calcified tissues have been observed and found to give good Raman spectra with an argon ion laser source. Walton et al. 219 compared these spectra with data from infrared spectroscopy. The quality of the Raman spectra was superior to that of the infrared ones in the case of adult human bone, but the situation was reversed for the examination of collagen. The Raman spectrum of bone was dominated by a 955 cm-1 band associated with the symmetric O--P--O stretching in the calcium phosphate. Bands at 1451 and 1417 cm-1 were characteristic of inorganic carbonate. Methylene bending modes of proline and hydroxyproline also scattered in the 1451 cm-1 region. Other band assignments were also given for amide I, II, and III, and phenylalanine benzene rings. III. R E S O N A N C E - R A M A N APPLICATIONS A. Carotenoids and Plant Pigments
One can obtain resonance enhancement in Raman spectra by using an excitation frequency close to an absorption peak of the scatterer32° Carotenoids and vitamins A show a pronounced effect,221and the shape of their excitation profiles 222 allows one to observe selectively a specific pigment in a complex system such as plant tissue. 223The vibrational modes of earotenoids involving the skeletal C = C , C--C, and C~-N stretching deformations, which most effectively modulate the ehromophore, are the only ones to show resonance enhancement?24 Gill et al. 223 have presented resonance Raman spectra for live carrot root and live tomato fruit (Fig. 15). Lutz and Breton 2~5 have studied the resonance Raman spectra of spinach chloroplasts and grana. They ,showed that with variations of excitation wavelength individual enhancement of chlorophyll a, chlorophyll b, and carotenoid contributions was observed. They also presented spectral changes from chlorophyll association states in the chloroplasts. They used argon laser excitation, and for some experiments a helium-cadmium continuous laser (441.6 nm). B. Enzymes
Salmeen et al326 studied the resonanee-Raman spectra of cytochrome c oxidase as the solubilized form and in APPLIED SPECTROSCOPY
141
1527 LIVE {ARROI IEOI
L~
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t ~
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II~6~158
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I 1,300
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._ I ___ I .... t 1,200 I,I00 1,000
900
cm-Z
a LIVE TOMATOFRUIT
15225
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cytochrome c oxidase displayed anomalous redox behavior. The spectra of cytochrome c at 441.6 nm excitation were very different from those of Spiro and Strekas22V, 2~s which were obtained with laser excitation (514.5 nm) near the a- and #-bands. These differences are related to the differences in the electronic bands in resonance with the incident laser frequency. Spectra of oxyhemoglobin and deoxyhemoglobin excited at 488 n m 229 resemble more closely the hemoprotein spectra that Salmeen et al. ~26 have observed with 441.6 nm excitation. Carey and Schneider 23° recorded resonance Raman spectra of cinnamoyl and a-toluyl acyl derivatives of a-chymotrypsin. T h e y identified bands associated with the aromatic portion of the acylating groups, bands which could be distinguished in a cinnamoyl derivative from those associated with the ethylenic residue. T h e y also observed spectral differences in the acyl enzyme relative to substrate and product. The spectral differences, which were probably caused b y changes in vibrational modes of substrate bonds due to specific interaction with the active site, provided another approach to the study of the mechanisms of enzyme activity.
965
C. Hemoglobin, Cytochrome c, and Related Sbustances
BOTTLED TOMATO SAUCE 1522
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LYCOPENE IN n-HEXANE I
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l
I
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1,450
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b FiG. 15. Vibrational fundamentals in the resonance-enhanced Raman spectra of: a, A-carotene in live carrot root, in canned carrot juice and the n-hexane solution of the pure all-trans pigment (Sigma) (excited at 488 nm); b, lycopene in live tomato fruit, bottled tomato sauce (the 1527 line of the canned carrot juice spectrum was taken with the gain reduced by a factor of 2), and solutions of lycopene extracted from bottled tomato sauce in an n-hexane solution (excited at 514.5 nm). Excitation by argon ion laser, incident power ~ 100 mW. Grazing angle reflection geometry: the electric vectors of incident and of analyzed scattered light are both perpendicular to the scattering plane. Spectrometer: 75 cm Czerny-Turuer double monochromator, digital photon counting detection, integration time 1 sec, scanning speed 20 cm-1/min. 223With permission of the editors of N a t u r e and the authors. electron transport particles. They used laser excitation near the Soret band. As in the spectra of other hemoproteins--for example, cytochrome c - - t h e shape and intensity of several bands changed with variation of the oxidation state. One of the hemes of the solubilized 142
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Spiro and Strekas 231 recorded the resonance-Raman spectra of hemoglobin and of cytochrome c.~2s The spectra exhibited a rich pattern of bands from 600 to 1700 em -1, arising from vibrational modes of the heme chromophores. Resonance-Raman spectra of hemoglobin and cytochrome c contained prominent bands that exhibited i n v e r s e polarization2~7; i.e., the polarization vector of the incident radiation was rotated through 90 ° for 90 ° scattering giving infinite depolarization ratios. These authors have discussed an antisymmetric molecular-scattering tensor required b y this phenomenon. Yamamoto et al. ~32 did a comparative study of the resonance-Raman spectra of several hemoproteins with excitation in the Soret region: cytochrome b~, metmyoglobin, oxyhemoglobin, deoxyhemoglobin, methemoglobin, and methemoglobins. This study revealed features that characterize the valence and spin state of the iron. Reduced (ferrous) hemoproteins always displayed the strongest band of their spectra between 1356 and 1361 cm-L Oxidized (ferric) proteins showed their strongest band between 1370 and 1378 cm-L E v e r y system studied showed a band in the conjugated double bond stretching region, with the principal component at 1584 and 1566 cm -~ for the low and high spin configurations, respectively. The data for oxyhemoglobin-strongest band at 1375 cm -1 and the 1584 cm -~ band domination of the double bond stretching region--support the assignment of a low spin ferric structure to the iron ion. Woodruff and Spiro 23~ devised a cell which holds circulating solutions. The device allows the use of high exciting energy with absorbing samples, while simultaneously permitting temperature control and simple scattering geometry. Spectra of oxy cobalt hemoglobin,
deoxy cobalt hemoglobin, and mixed cobalt hemoglobin were recorded in such a cell. Brunner and Sussner 284 reported that resonanceRaman spectra of intact red blood cells are actually the spectra of the hemoglobin molecules inside the cells. These workers also studied the spectra of oxy-, deoxy-, met-, and cyanomethemoglobin, and of protoporphyrins IX and chlorohemin in solution. None of the Raman lines of pyrrole235 was found. Most of the Raman lines arise from vibrational modes involving the C = C , C--C, and C = N bonds of the conjugated ring of the heme group. Raman lines were also found below 500 cm-~ due to Fe--N-vibrations in the hemoglobins and in chlorohemin. The line at 572 cm-1 in oxyhemoglobin corresponds to the vibration of the ligand against the iron. Loehr et al. 236 found that the oxygenation of hemocyanin produces resonance-Raman peaks at 742 and 282 cm-~. The former peak which was in resonance with a 575 nm charge-transfer band shifted to 704 cm-~ when ~sO2 was substituted for ~602. The bound oxygen was in the form of peroxide (O22-). The 282 cm -1 peak which was in resonance with a 340 nm optical transition was insensitive to isotopic substitution, suggesting that the 282 cm-~ peak corresponds to a vibration involving the magnetically coupled Cu(II)..Cu(II) centers. Mayer et al. 23~ recorded the resonance-Raman spectra of cyanocobalamin (vitamin Bi~) and dieyanocobinamide. These substances displayed essentially identical spectra in spite of substantial chemical differences, thus indicating that only those modes associated with their common corrin ring system were resonance-enhanced.
serum albumin. The Raman data were consistent with the results of other studies suggesting that bound methyl orange is in a medium of lower dielectric constant than water and the view that the SO3- interacts with a charged region of the protein. The data also indicate that strong interactions do not occur with the dimethylamino group or the azo group and that positive charge(s) or water molecules or both are asymmetrically distributed about the SOs-. The bound phenyl groups are not twisted out of the aqueous conformatJ.on. Carey et al. 241 obtained resonance-Raman spectra of 2,4-dinitrophenyl haptens free in solution and complexed with homologous, specifically purified rabbit antibodies. Many spectral changes occurred on complex formation. These resulted from short range interactions and the spectra contained much information about the complex. Binding by antibody of azo haptens caused them to twist about at least one of the C--N bonds of the azo group. The hapten 1-hydroxy-4-(2,4-dinitrophenylazo)-2,5-naphthalenedisulfonic acid existed in solution in a form where the naphthyl and phenyl rings were not coplanar. On binding, a fraction twisted about the azo group in the direction of greater planarity. However, the hapten 1-hydroxy-2-(2,4-dinitrophenyL azo)-3,6-naphthalenedisulfonic acid, when unbound, had its aromatic rings essentially coplanar, but they twisted out of this conformation on binding. Infrared spectra were also used in this study to obtain structural information. The authors suggested other possible haptens for future studies, the NO2 group not being an optimal choice.
D. Visual Pigments and Related Substances
IV. CONCLUSION
Rimai et al. TM reported the detection of resonanceenhanced vibrational Raman scattering from whole bovine retinas at temperatures between - 7 0 and -85°C. The observed spectra from dark-adapted retinas were contributed only by the visual pigments. The strongest line, at 1551 cm-1, was contributed by the ethylenic C-~C stretching mode of retinal. Results on Raman spectra of the model system retinylidene hexylamine in acidified ethanol solution showed this mode at 1560 cm-~, supporting the hypothesis that the pigment in the retina is bound as a protonatcd Schiff base. Gill et al. 238 recorded resonance-Raman spectra of three retinal isomers: the 11- and 13-cis isomers and trans-retinal. They indicated the usefulness of the spectra for monitoring isomerization reactions. Rimai et al. 239 studied the resonance-Raman spectra of retinals (trans, 9-cis, 13-cis), retinols (trans, 13-cis), and trans-retinoic acid in octanol solution. The terminal group was identified by the frequency of the line at 1580 to 1590 cm-~, while the isomer was uniquely characterized by the lines in the 1100 to 1400 cm-1 region.
The usefulness of infrared spectroscopy for biochemical application will continue into the future. It is safe to presume that, in particular, the field of porphyrin chemistry will benefit from such applications, m The introduction of Fourier transform instrumentation is a big step in the right direction. Also, the ability to obtain with Fourier transform spectroscopy complete infrared spectra of biochemical molecules in aqueous systems by subtracting the spectrum of water appears to be a major advance. Raman spectroscopy has given much new information that complements knowledge available from infrared spectroscopy. In particular, new information has come from the fields of protein and polypeptide chemistry (including enzymes) ; the chemistry of nucleic acids and their constituent moieties (nucleosides, nucleotides, etc.); the chemistry of carbohydrates and steroids; and the chemistry and function of biological membranes and their constituents. Much of the information gained has been conformational. The literature on resonance-Raman applications in biochemistry is growing rapidly, and much new information should be forthcoming on a variety of molecules. The ability to observe selectively a specific pigment in a complex tissue22~is a significant advance in biochemical application. There is so much still to be learned concerning enzymes and how they work that this area also looks very promising. The same can be said for work on
E. Protein-Ligand and Antibody-Hapten Interactions Carey et al. 2~° demonstrated the feasibility of using resonance-Raman spectroscopy to obtain information about protein-ligand interactions. They obtained vibrational spectra for methyl orange bound to bovine
APPLIED SPECTROSCOPY
143
r e s p i r a t o r y p i g m e n t s , e.g., v a r i o u s h e m o g l o b i n s , c y t o c h r o m e c, a n d h e m o c y a n i n s . Also p r o m i s i n g as a p p l i c a t i o n s of r e s o n a n c e - R a m a n s p e c t r o s c o p y are s t u d i e s on protein-ligand and antibody-hapten interactions. This is a n e x c i t i n g p e r i o d w h e n one c o n t e m p l a t e s t h e i m p a c t of s p e c t r o s c o p y g e n e r a l l y in t h e b i o c h e m i c M field. h o p e f u l l y , t h e e x c i t e m e n t will c o n t i n u e for a long t i m e . 1. F. S. Parker, Applications of Infrared Spectroscopy in Biochemistry, Biology, and Medicine (Plenum Press, New York, 1971), and references therein. 2. R. P. Bauman and C. Clark (Conference Co-chairmen) and others, "Biological Applications of Infrared Spectroscopy," in Ann. N. Y. Acad. Sci. 69, Art. 1, (1957), and references therein. 3. C. N. R. Rao, Chemical Applications of Infrared Spectroscopy (Academic Press, New York, 1963). 4. J. T. Edsall, J. Chem. Phys. 4, 1 (1936); J. Chem. Phys. 5,508 (1937). 5. J. T. Edsall, J. W. Otvos, and A. Rich, J. Am. Chem. Soc. 72,474 (1950). 6. D. Garfinkel and J. T. Edsall, J. Am. Chem. Soc. 80, 3807 (1958). 7. D. Garfinkel and J. T. Edsall, J. Am. Chem. Soc. 80, 3818 (1958). 8. D. Garfinkel and J. T. Edsall, J. Am. Chem. Soc. 80, 3823 (1958). 9. D. Garfinkel, J. Am. Chem. Soc. 80, 3827 (1958). 10. G. Herzberg, Molecular Spectra and Molecular Structure: Infrared and Raman Spectra of Polyatomic Molecules (Van Nostrand, New York, 1945). 11. E. B. Wilson, J. C. Decius, and P. C. Cross, Molecular Vibrations (McGraw-Hill, New York, 1955). 12. N. L. Alpert, W. E. Keiser, and H. A. Szymanski, IRTheory and Practice of Infrared Spectroscopy (Plenum Press, New York, 1970), 2rid ed. 13. W. J. Potts, Jr., Chemical Infrared Spectroscopy, Vol. I, Techniques (Wiley, New York, 1963). 14. M. Davies, Ed., Infrared Spectroscopy and Molecular Structure (Elsevier, New York, 1963). 15. N. B. Colthup, L. H. Daly, and S. E. Wiberley, Introduction to Infrared and Raman Spectroscopy (Academic Press, New York, 1964). 16. H. A. Szymanski, Ed., Raman Spectroscopy, Theory and Practice (Plenum Press, New York, 1967). 17. G. J. Thomas, Jr., "Infrared and Raman Spectroscopy," in Physical Techniques in Biological Research, G. Oster, Ed. (Academic Press, New York, 1971), ¥ol. I, Part A, 2nd ed. Chap. 4. 18. R. J. Obremski, Introduction to Raman Spectroscopy (Beckman Instruments, Inc., Fullerton, Calif., 1971). 19. K. D. M611er and W. G. Rothschild, Far-Infrared Spectroscopy (Wiley-Interscience, New York, 1971). 20. M. C. Tobin, Laser Raman Spectroscopy (Wiley-Interscience, New York, 1971). 21. S. K. Freeman, Applications of Laser Raman Spectroscopy (Wiley-Interscience, New York, 1974). 22. N. J. Harrick, Internal Reflection Spectroscopy (Wiley, New York, 1967). 23. J. A. Schellman and C. Schellman, "The Conformation of Polypeptide Chains in Proteins," in The Proteins, H. Neurath, Ed. (Academic Press, New York, 1964), Vol. II, 2nd ed., Chap. 7. 24. F. R. Quinn and F. A. Bettelheim, Biochim. Biophys. Acta 59,544 (1963). 25. C. B. Baddiel, J. Mol. Biol. 38,181 (1968). 26. J. E. Katon, J. T. Miller, Jr., and F. F. Bentley, Carbohyd. Res. 10, 505 (1969). 27. J. E. Katon, J. T. Miller, Jr., and R. R. Ferguson, Technical Report AFML-TR*68-169, Air Force Materials Laboratory, Wright-Patterson Air Force Base, Ohio, 1968. 28. T. S. Hermann, Appl. Spectrosc. 23,461,473 (1969); with 144
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