VOL. 14, 379-391 (1975)

BTOPOLYMERS

Raman Scattering of Collagen, Gelatin, and Elastin BRUCE G. FRUSHOUR and JACK L. KOENIG, Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106

Synopsis The ltaman spectra of collagen, gelatin, and elastin are presented. The Raman lines in the latter two spectra are assigned by deuterating the amide N-H groups in gelatin and by studying the superposition spectra of the constituent amino acids. Two lines appear a t 1271 and 1248 cm-1 in the spectra of collagen and gelatin that can be assigned to the amide I11 mode. Possibly, the appearance of two amide I11 lines is related to the biphasic nature of the tropocollagen molecule, i.e., proline-rich (nonpolar) and proline-poor (polar) regions distributed along the chain. The melting, or collagen-togelatin transition, in water-soluble calf skin collagen is studied and the 1248-cm-I amide I11 line is assigned to the 31 helical regions of the tropocollagen molecule. Elastin is thought to be mostly random and the Itaman spectrum confirms this assertion. Strong amide I and I11 lines appear a t 1668 and 1254 cm-', respectively, and only weak scattering is observed a t 938 cm-1. These features have been shown to be characteristic of the disordered conformation in proteins.

INTRODUCTION Collagen and elastin are the principal structure proteins of many vertebrate and invertebrate species and are located in most structural t'Issues including tendons, the walls of blood vessels, skin, and bone. Gelatin is the thermal denaturation product of collagen. Unlike the globular proteins that usually function in organisms as discrete units or interacting subunits, collagen and elastin have fibrous tertiary structures and combine with other macromolecules to form the structural tissues, which support the mechanical stresses of the organism. I n tendons, for exampie, the collagen fibers run parallel to the major strcss axis. Ultimately, the gross mechanical properties of the collagenous tissue must be derived from the conformation of the collagen molecule. Raman spectroscopy offers one approach to studying the chemical and conformational changes of the collagen molecule. Most of the conventional techniques for studying the conformations of biopolymers require their solubility, usually in water. However tendons or other collagenous tissues are not soluble (although water-soluble collagen can be prepared). The solid-phase sampling techniques available with Raman spectroscopy should cnable one to study the conformations of the collagen rnolccule in intact tendons and other tissues.2 Important chemical and conformational changes of collagen fibers associated with the phenomena of swelling, straining, and heating can be studied. 379

@ 1975 by John Wiley & Sons, Inc.

380

FIZUSHOUR AND KOENIG

The Raman spectra of collagen in intact OX tibia (bone)3and tooth dentyne4 have been reported previously. Only a few lines were assigned and the signal-to-noise ratio was low. Previous attempts to obtain the spectrum of purified collagen and gelatin with 4SOO-8 laser excitation in this laboratory failed. The cross section of a tendon reveals collagen fibers approximately 20 pm in diameter embedded in a ground substance consisting of mucopolysaccharides and other molecules. Closer examination of a collagen fiber reveals fibrils 5000 8 thick separated by 200 8. These fibrils consist of tropocollagen molecules packed in a quarter-staggered array.5 Tropocollagen molecules have a rodlike shape of dimensions 15 x 3000 8 and are formed by twisting togc.ther three scparatc polypeptide chains into a three-stranded coiled-coil with a right-handed screw sense.5 The molecular weight of the tropocollagen molecule is 300,000 g/mol, i.e. about 100,000 g/mol for each chain. The t h r w chains, or a chains, appear to be very similar. The amino acid sequence of the a1 chain fragments arid the staining pattern of the tropocollagen fibrils observed in the electron microscope reveal regions along the chains rich in nonpolar amino acids such as proline, hydroxyproline, and alanine and regions rich in polar amino acids such as lysine, glutamic acid, and aspartic acid.5 Except for the N-tcrminal scction of the chains, which are about 10-15 residues long, every third residue of the chains is glycine. Therefore, a model for thc regions of the chain rich in nonpolar amino acids would bc Gly-Pro-X, where X may bc alanine or some other nonpolar residue and the corrwponding model for the polar regions would bc Gly-X-1'; X or Y being nonpolar amino acids such as glutamic acid and lysinc.'j From X-ray diffraction studies of the collagen fibers and sequcntial polypeptide collagen models it has been dctcrmined that the nonpolar (proline-rich) regions of the a chains adopt the Iefthanded 31 helical conformation. The X-ray data of the collagen fibers requires that thc three a chains be twisted about a common axis resulting in a triple-stranded c~iled-coil.~Since each strand of the coiled-coil must be a 31 hclix, the nonpolar regions of the three a chains must be in register. The conformation of thc polar regions of the a chains has not been determined and is not thought to be highly ~ r d e r e dit . ~ Therefore, two diff ercnt secondary conformations arc probably distributed along thc polypeptide chains of collagcm Several regions of the Raman spmtrum, including the amide I and I11 normal models8-11 may reflect this distribution of conformations. Elastin, another fibrous protein, occurs in collagcncous tissue when elastic properties are required. l 2 Examples include the aorta, skin, arid ligaments. A particularly pure source of elastin is bovine Ziyamentum nuchae, the neck ligament of grazing cows. Elastin appears to have no well-defined secondary structure, though Mammi e t al. l 3 have reported that native elastin may contain up t o 10% a-helix. A unique feature of elastin is the presence of the amino acids dcsmosinc arid isode~mosine.'~ These amino acids are tetrasubstituted aromatic pyridinium rings arid serve as cross-links in the insoluble native elastin.

RAMAN SPECTRA OF COLLAGEN, GELATIN, ELASTIN

381

I n this initial study we present the Raman spectra of collagen, gelatin, and elastin. Since the amino acid composition of collagen is diff erent from those of the globular proteins previously studied by Raman spectroscopy, the superposition spectra of the constituent amino acids and the spectra of deuterated gelatin were used in assigning the Raman lines in the collagen spectrum. We have also studied the collagen-to-gelatin transition in water-soluble calf skin collagen.

EXPERIMENTAL Collagen from bovine Achilles tendon and calf skin was purchased from Sigma Chemical Company, St. Louis, Mo. The calf skin collagen had been water-soluble during the purification process but was insoluble when received. A solution of calf skin collagen containing 6 mg collagen/l ml 0.075 citrate buffer a t p H 3.6 was purchased from the Worthington Biochemical Company, Freehold, N.J. Purified calf skin gelatin was obtained from the Eastman Kodak Company, Rochester, N.Y. Elastin from bovine Zigamentum nuchae was obtained from the P-L Biochemical Company, R4ilwaukee, Wise. and from Worthington Biochemical Company. There were no measurable differences in the Raman spectra of the two samples. 5% solutions of gelatin were obtained by dissolving the protein in water at 50°C, filtering the solution through a 0.80-pm Rllillipore filter, and allowing the solution to gel inoa liquid sample cell for the Raman spectrometer. The laser beam, 5145.3 A, was passed up the axis of the tube through the gelatin and the Raman scattering was collected a t right angles to the laser beam. The calf skin collagen solution was too dilute (O.6yO), and contained too much citrate buffer for the Raman experiments. The as-received solution was dialyzed against water a t pH 3.5 t o remove the buffer and then concentrated by a careful freeze-drying and thawing technique until a n extremely viscous 2% solution was obtained. The concentrated solution was filtered through 5-pm and 1.2-pm Millipore filters before recording the spectrum. Gelatin was deuterated by dissolving 1 g of the protein in 100 ml of warm DzO. After setting for 24 hr the solution was freeze-dried and the gelatin redissolved in fresh DzO to a filial concentration of 10%. The circular dichroism (CD) spectra were recorded on a Jasco 5-20 recording spectropolarimeter. The Raman spectrometer has been described ~lsewhere." A Spectra-Physics Model 165 argon ion laser tuned to 5145.3 A provided the Raman excitation. For specific power levels see the figure captions.

RESULTS Raman Spectra of Collagen and Gelatin The Raman spectrum obtained from the collagen of bovine Achilles tendon appears in Figure 1A. A similar spectrum could be obtained from

FRUSHOUR. AND KOENTG

382

1451

i

1248

1271

A

1006

o,,

876

/

w

A

1 1248

1451

,

'1800

'1600

1271

'1400

A

'1200

'1000

'800

'600

'400 CM-'

Fig. 1. Itaman spectra of collagen from bovine -4chilles tendon ( A ) and calf skin gelatin ( B ) ;slit width, 8 cni-'; scan rate, 10 cm-'/min; laser power a t sample, 300 mW in ( A )and 600 mW in ( B ) ;time constant, 10sec.

the insoluble calf skin collagen. The signal-to-noise ratio in these spectra is much higher than in the spectra of ox tibia3 and dentyne4 recorded previously, and many more lines can be observed. Raman studies of the polypeptides and globular proteins indicate that the amide I and I11 regions of the Raman spectrum are very sensitive to secondary structure of the polypeptide chain. Two lines appear in both the amide I and I11 regions of the collagen spectrum in Figure 1A a t 1670 and 1642 em-' and also 1271 and 1245 cm-l, respectively. The spectrum of gelatin, Figure lB, is very similar to the spectrum of collagen. One is tempted to associate the appearance of two lines in the amide I and I11 regions with the polar and nonpolar regions of the polypeptide chains in collagen. However, alternative explanations of this apparent splitting must also be examined. The amino acid composition of collagen is quite different from those of the globular proteins reported in previous Raman investigations, and line assignments based upon these studies may not be applicable to collagen. Therefore, the amide I11 region of the collagen spectrum may contain side-chain Raman lines not present in the spectrum of the globular proteins. We have investigated this possibility by comparing the gelatin spectrum with the superposition spectrum of the constituent amino acids a technique demonstrated by Lord and YuI5for several globular proteins. I n Figure 2, the Raman spectrum of gelatin, which is very similar to the collagen spectrum, is compared to a spectrum of an amino acid solution

383

RAMAN SPECTRA OF COLLAGEN, GELATIN, ELASTIN

pH 13

11100

I1600

I1400

IIn,

llooo

ll00

1600

1400

CM-l

Fig. 2. The Raman spectra of calf skin gelatin and an amino acid mixture. The concentrations of the amino acids in t,he mixture correspond to the amino acid composition of the gelatin.

prepared so that the concentration of the individual amino acids corresponds t o the amino acid composition of gelatin.16 The superposition spectra were recorded a t both pH 2 and 13 so that the extraneous Raman lines of the amino and carboxyl groups not on the side chain could be identified. Most of the differences between the two superposition spectra can be traced t o the ionization of the carboxyl groups; the Raman lines of the NH? and NH3f groups are very weak.'? Only weak scattering appears from 1300 t o 1200 em-' in the two superposition spectra, as indicated by the dashed lines in Figure 2, suggesting that the two lines appearing a t 1271 and 124s em-' in the spectrum of gelatin do not arise from the side chains. However, they still might correspond t o the vibrations of the proline and hydroxyproline residues, apparent in this region of the Raman spectrum of poly-L-proline and poly-L-hydroxyproline. 18*19 These two amino acids comprise approximately one-fourth of the amino acids in collagen. They have no amide hydrogen and therefore no amide I11 mode, which involves the amide NH in-plane bending motion. These lines, if they were also present in the collagen spectrum, would not be expected to shift upon deuteration of the amide N-H. The spectrum of deuterated gelatin appears in Figure 3. Two

FRUSHOUR AND KOENIG

384

1464

A

1664

I

1700

I

I

1500

1

I

1300

I

I

1100

I

I

900

1

-1 CM

Fig. 3. Raman spectrum of deuterated gelatin, 10% concentration in L),O; slit width, 6 cm-*; scan rate, 10 cm-'/rnin; power, 600 mW a t sample; time constant 10 sec.

weak lines appear in the amide I11 region a t 1274 and 1247 em-', which probably corresponds to the strong lines appearing a t 1271 and 1248 em-' in the collagen and gelatin spectra of Figure 1. Also, two lines of medium intensity appear a t 993 and 966 em-' in the spectrum of deuterated gelatin not present in the spectrum of collagen and are assigned to the amide 111' modes (deuterated analog of the amide I11 mode). Previous deuteration studies of proteins and polypeptides show a shift of the amide I11 mode to 900-1000 em. 1-15 On the basis of the superposition spectra and the deuteration of gelatin, we conclude that the lines appearing a t 1271 and 1248 em-' in the spectra of collagen and gelatin can be assigned to the amide I11 mode. Possibly these two lines can be associated with the polar and nonpolar regions of the collagen polypeptide chains. This mill be discussed later. Table I includes the Raman lines of all the spectra pertaining to collagen. When possible, the,assignments are included. The superposition technique was extended to include a mixture of the amino acids tyrosine, histidine, phenylalaninc, proline, and hydroxyproline. These amino acids have either aromatic or saturated rings in their side chains and arc strong Raman scatterers. Some of the more distinctive side-chain lines in the collagen spectrum include phenylalanine (1037, 1006 em-'), proline (925,860 cm-l), and hydroxyprolinc (880 cm-I). When calf skin collagen is heated above 37°C the helical regions of the tropocollagen molecule melt and become random. This transition is complctely reversible only under certain conditions. The conformation of the polypeptide chains in gelatin, the thermal denaturation product of collagen, is thought to be largely random, though short regions of the triple helix still remain and serve as cross-linlis.'6 As mentioned before, the Raman spectra of both collagen and gelatin appear in l'igure 1 and are very

RAMAN SPECTRA O F COLLAGEN, GELATIN, ELASTIN I

I

I

I

I

I

385

I

1248

1453

1453

1500

1400

1300

-1 1200 CM

Fig. 4. Raman spectra of native and thermally denatured calf skin collagen. ANative, 2% concentration a t pH 4.0,25°C; instrument conditions same as Figure 1A except power is 1 W. B-Denatured, same as above but heated at 70°C for 2 hr and cooled to 25°C.

similar. Much fluorescent background was encountered when recording the collagen spectrum; the sample had to be irradiated with 300 mW of power for 6 hr before the fluorescence decayed to a n acceptable level. The irradiated region of the collagen sample may have been melted (heated above 37°C) by the laser beam, implying that both spectra in Figure 1 might be of largely disordered chains. Since sample heating and fluorescence are much reduced in solution, we decided to examine native and thermally denatured solutions of collagen. The Raman spectrum of a 2% solution of calf skin collagen appears in Figure 4A. I n the amide I11 region of the Raman spectrum, lines appear a t 1248 and 1274 em-], as in the spectra of collagen discussed previously. The background scatter below 1000 cm-', characteristic of dilute solutions, prevented the observation of the Raman lines a t lower frequency. More concentrated solutions were too viscous to handle. The CD spectrum of this sample obtained a t

1668s 1636 s sh

Collagen (B.A.T.)

1670 s 1642 s sh

1464 s sh 1-151s

1422 m 1399m 1389 m 1347 m 1320 m

1271s

1248s 1211w 1198w 1182 w

1165 w

1128w

1101w 1084w

1464 s sh 1451s

1422 m

1392m 1343m 1314 m

1271s

1248 s 1211 w

1178 w

1161 w

1128 w

1101w 1087w

1566 w

1608w

Gelatin (10% Aqueous Solution)

1111m 1088 m

1188w

1211w

1245 w

1353 s 1323 m

1415 vs

1450 s

1611 m 1589 m

Mixture of All Amino Acids pH 13

1091w

1118111

1188w

1211 m

1091 m

1188m

1211 m

1238 w

1274 w

1271 w 1235 m

1330 m 1320 m

1396 s

1451 s

1483 w

1604 m 1585 m

Mixture of Aromatic Amino Acids p H 13

1353m 1324 m

1438s

1460s

1611m 1589 m

1746s

Mixture of All Amino Acids PH 2

1088m

1111m

1145 w

1178 w

1350 m 1320 m

1408 s

1457 s

1576 m

1601 m

Mixture of Nonaromatic Amino Acids pH 13

DzO

Gelatin (l0yo in

1105

1135 w

1185 w

1247 w 1211 6(DzO)

1274 w

1347 m 1330 m

1415 m

1464 s

1611 w

1664 s 1645 s sh?

TABLE I Raman Lines in ColIagen and Related Spectra

+

u(C-N)

u(C-N)

NHj

TYr

h i d e 111 HYPro, TYr

Amide I11

rdCH3, CH2), rt(CHa, CHz), 6 (Cs-H

6(CH3, CH2) 6(CH3, CHz) in. pl. bend of carboxyl OH &.,(coo-)

Phe, Tyr Pro., Hypro

u(C=O) Amide I Amide I

Assignment

Q,

cu oa

969 sh

942 s

925 s

890 w 880 s

863 s

818 m

769 w

625 w

572 w 536 w

966 w

938 m

921 m 918 m

890 w 876 m

856 m

821 w

769 w

622 w

568 w 533 w

543 s 518 m 447 w

629 w 593 w

783 m

856 vs

414 m

741 s 650 m 629 m 593 w 575 w 522 m 504 s

825 vs

870 vs

918 m

969 m

1006 m

1044 s

345 w

479 m 447 m

646 w 629 w

786 w

859 s

873 s

918 s

1006 s 982 m

1034 m

1051 w

540 w 522 w

593

783 w 668 w

866 s

908 s

942 s

1024 w

407 w

575 w 540 w

759 w Br

814 m

856 s

884 s

925 s

942 s

1006 s 993 m 966 m

1057 w

Pro

Pro EIypro

Phe

u(C-C) u(C-C) u(C-C) u(C-C) v(C-C)

of Hypro ring of residues of Pro ring of residues of backbone

Phe Amide 111‘ Amide 111’ v(C-C) of residues u(C-C) of protein backbone v(C-C) of Pro ring

bend of carboxyl OH Pro 0. pl.

Key to abbreviations: s(strong), m(medium), w(weak), sh(shoulder), vw(very weak), v(stretching coordinate), &(deformationcoordinate), -yw(wagging coordinate), -yt (twisting coordinate), Tyr(tyrosine), Phe(phenylalanine), Trp(tryptophan), Pro(proline), Hypro(hydroxyproline), B.A.T. (Bovine Achilles tendon).

396 w

1006 m

1006m

425 w

1006 m 986 w

1037 m

1037 m

907 vs

1037 w

1064 w 1051 w

1067 w

388

FRUSHOUR AND KOENIG

4 0 I

2

2

X n

&

0

-2

1 200

I

1

1

I

220 WAVE LENGTH,

240

MP

Fig. 5 . Circular dichroism spectra of native and thermally denatured calf skin collagen. A-Native collagen, 25OC. B-Denatured, heated a t 70°C for 2 hr then run a t 25OC.

25°C before and after irradiation by the laser beam was identical and appears in Figure L4. The spectrum exhibits a positive band with weak molar ellipticity a t 218 nm characteristic of native collagen.20 After recording the Raman spectrum, the sample tube was immersed in a water bath and heated a t 70°C for 2 hr. A small aliquot of the heated solution was placed in a CD cell and diluted to the appropriate concentration a t 25°C. The CD spectrum, seen in Figure 5B now displays a sloping line of negative ellipticity characteristic of a randomly coiled polypeptide chain. The denaturation is reflected in the Raman spectrum by small but definite changes, seen in Figure 4. The stronger of the two amide I11 lines has shifted from 1248 to 1251 em-' upon denaturation and the amide I11 region appears to have broadened slightly. Also the intensity ratio of the stronger amide I11 line to the 1453-em-' line assigned to the methyl and methylene deformation modes has decreased from 1.3 to 1.2. The 1453-em-' line appears to be a suitable internal intensity standard and has been used by us in previous Raman studies for this purpose. Recent conformational studies with poly-L-lysine are relevant to the interpretation of the collagen spectra. Tiffany and I