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Fourier-transform Raman spectroscopy of mammalian and avian keratotic biopolymers
SPECTROCHIMICA ACTA PART A
ELSEVIER
Spectrochimica
Fourier-transform
Acta Part A 53 (1997) 81-90
Raman spectroscopy of mammalian avian keratotic biopolymers W. Akhtar”,
Vhemistry
H.G.M.
and
Edwards”,*
and Chemical Technology, University of Bradford, Bradford BD7 IDP, UK
Received 27 March 1996; revised 23 May 1996; accepted 24 May 1996
Abstract The FT-Raman spectra of mammalian and avian keratotic biopolymers have been recorded, including bull’s horn, cat’s claw, bird’s feather quill, pheasant’s beak and compared with the hard keratinous tissue, human nail and callus. Although there were similarities in all the spectra, particularly in the v(CH) stretching region, the 1450-l 100 cm- 1 region exhibited some differences ascribed to intramolecular skeletal backbone conformational changes. Of particular significance for human, mammalian and avian samples in the 1000-400 cm _ ’ wavenumber region were differences in the structurally important v(SS) and v(CS) bands, near 500 cm-’ and 630 cm- I, respectively. The amide I and III modes near 1650 and 1250 cm- ’ respectively, demonstrate that the mammalian keratins studied exist predominantly in the a-helical conformation, whereas the avian keratins adopt the p-sheet structure as the dominant conformation. Keywords:
Avian keratotic biopolymers;
FT-Ramam
spectroscopy; Mammalian
1. Introduction Keratins are naturally occurring proteins which owe their rigidity and strength to sulphur-sulphur intramolecular cross-links between cysteine amino acid residues and to extensive intramolecular hydrogen bonding. Keratotic structural tissue is found in reptiles, birds and mammals and may also serve as a barrier to environmental stress. Keratins are the principal constituent of the outermost layer of human and animal skins and in appendages such as horns, hooves, hair and feath-
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biopolymers
ers. Whereas rhinoceros horn represents a primitive fibrous keratin, reptilian or squamate scales are highly sophisticated adaptations of different keratins in the same sample which fulfill specific purposes; the soft, flexible hinge of snake skin is mainly a-helical keratin whereas the hard scales are composed of layers of the p-sheet conformation. Some tissues have evolved from hair, e.g. animal horns, and their keratotic composition is quite similar. In some earlier studies from our laboratories, the keratotic tissue in the skins of reptiles and mammals, including three species of snake [1,2] and pig [3], were characterized using Raman spectroscopy and compared with human stratum
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W. Akhtar, H.G.M. Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
Table 1 Amino acid composition’ of mammalian and avian keratins Residue
Human stratum corneumb
Human nail”
Bird’s featheP
Bull’s horn’
alanine arginine aspartic acid half cystine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tyrosine valine
7.1 4.5 9.8 2.9 15.8 11.6 1.6 4.6 9.3 5.7 0.5 3.4 2.9 7.2 5.4 2.0 5.7
6.5 6.7 8.9 7.4 15.2 6.6 0.9 3.9 9.9 4.5 0.8 2.2 4.1 8.7 5.0 2.8 5.9
8.7 3.8 5.6 7.8 6.9 13.7 0.2 3.2 8.3 0.6 0.1 3.1 9.8 14.1 4.1 1.4 7.8
8.1 8.7 8.7 4.2 15.1 7.1 0.8 4.5 10.7 4.0 0.6 2.6 3.4 7.9 4.8 2.7 6.0
a Measured as residues per 100 amino acids. b Ref. [lo]. ’ Ref. [ll]. d Ref. [12].
corneum [4,5], the hard outer layer of the epidermis which consists of keratin cells in a lipoidal matrix. Currently, knowledge of the keratotic structures of tissues is providing some useful information for the pharmaceutical therapeutic control of skin disease in humans and is essential for the understanding of transdermal drug delivery systems [6] and their operation in, for example, hormone replacement therapy using patches applied to the stratum corneum [7]. In this context, it has been shown that reptilian squamate keratins are not good models for human stratum corneum, despite their adoption for this purpose for drug diffusion studies [3]. Also, in the area of biomedical diagnostics, the application of Raman spectroscopic techniques [8,9] to the study of healthy and diseased skin tissue is providing novel information about the origins of skin afflictions and their treatment, e.g. psoriasis, skin cancer, melanomas and sunburn. When it is realised that almost 40% of all accidents in the workplace are skin-related and up to 10% of the population are affected by skin disorders such as acne, vitiligo, psoriasis and cancer, the importance of an understanding of keratin composition and
the spectroscopic changes therein for interpretation of Raman spectra on a diagnostic basis is of great significance. In this study, we report the Raman spectroscopic analysis of keratins from mammalian and avian tissues, including human samples, from which novel information is provided about molecular vibrational assignments and keratotic structure.
2. Experimental 2.1. Samples
In this work, representative samples of different keratotic tissues from mammals and birds were taken. A bull’s horn (Texas longhorn), cat’s claw (domestic cat) and pheasant’s beak represent mammalian and avian hard tissue; bird’s feather (pigeon) quill and cat’s fur represented softer tissue, and human nail and callus skin tissue samples were analysed to provide a comparison with the hard and soft keratotic samples from birds and mammals.
1448 1416
1338 1316 1266 1250 1207 1176 1156 1126 1096 1086 1061 1043 1030 1003 956 936
1385 1340 1318
1271 1251 1207 1174 1154 1126 1098 1084 1066 1051 1031 1003 961 934
1386 1341 1316 1296 1269 1249 1207 1175 1156 1126 1106 1088 1063 1048 1031 1003 956 937
1519
1585 1556
1657 1615
1449 1416
1652 1618 1608 1585 1552
1449 1422 -
1554 -
1670 1653 1615 -
2957 2931 2871 2855 2733
2955 2931 2871 2859 2731
Cat’s claw
1269 1245 1207 1175 1156 1127 1098 1086 1062 1043 1036 1003 958 938
1448 1416 1405 1385 1340 1315 1340 1318 1298 1269 1251 1208 1175 1157 1127 1107 1084 1062 1049 1032 1003 957 937
1449 1421
1651 1618 1606 1581 1554
1270 1241 1210 1174 1154 1126 1095 1085 1061 1045 1030 1003 956 935
1395 1342 1317
,^, - - _
1269 1243 1208 1174 1158 1126 1095 1080 1060 1049 1031 1003 959 937
1390 1340 1314
1449 1414
1523
1524 1508 1500 1449 1414
1667
2957 2935 2875 2853 2730
3064
Pheasant’s
of mammalian
1615 1606 1587 1556 -
quill
modes
1615 1608 1585 1550
1665
2960 2933 2880 2856 2726
2958 2933 2871 2856 2719
2953 2933 2874 2855
1668 1654 1616 1610 1587 1559 1533 1527 1519
3060
Feather
3061
tissue
of the vibrational
3060
Callus
descriptions
Cat’s fur
and approximate
3062
nail
(cm-‘)
3062
(cm-‘) Human
Wavenumber Bull’s horn
3062 3002 2955 2930 2873 2857 2730
wavenumbers
Table 2 FT-Raman
,_1
-
beak
_
keratotic
biopolymers
band
/_
,”
_1
R.
_.
.,,
,_,
in
,_
_,
,
_
v(CC) v(CC) v(CC); 6 (COH) v(CC) skeletal, tram conformation v(CC) skeletal, tram conformation v(CC) skeletal, random conformation v(CC) skeletal, tram conformation v(CC) skeletal, cis conformation v(CC) skeletal, cis conformation v(CC) aromatic ring p (CH,); b (CCH) olefinic p(CH,) terminal; v(CC) a-helix
I
6(CH) 6 (CHJ 6 (CH,) r(CN); s(NH). Amide III. cc-helix 6(CH,) wagging; v(CN). Amide III. Disordered
6 (CH,) 6[C(CH,),] symmetric 6 (CH,) symmetric
@NH) v(C = C) carotenoid v(C = C) carotenoid v(C = C) carotenoid 6(CH,) scissoring
v(CH) olefinic r(CH) aromatic v(CH,) asymmetric v(CH,) symmetric v(CHJ asymmetric r(CH,) symmetric “(CH) aliphatic (overtone and combination of 6(CH,) fundamentals) v(C = 0) Amide I p-sheet v(C = 0) Amide I a-helix v(C = C) olefinic v(C = C) olefinic v(C = C) olefinic s(NH), r(CN). Amide II
Assignment
and avian
8
494
509
146 644 622 603
852 829
411
v(SS) [gauche-gauche-gauche] v(SS) [gauche-gauche-gauche] 6 (CCC) skeletal
492 417
512
512
509
529 508
510
136 642 621 603
P (CM 6 (CCH) aromatic 6 (CCH) aliphatic p (CH,) in-phase p (CH,) in-phase V(G) [gauche] v(CS) [gauche] P (CH) wagging v(SS) [gaucheegauche-tram] 882 853 828 151 740 643 622
880 853 828 156 742 643 621
885 853 830 758 744 644 622
893 854 829
892 852 829 756 146 643 621
896 852 829 157 743 643 621
Assignment Pheasant’s beak
Feather quill
Callus tissue
Cat’s fur
Cat’s claw
Wavenumber (cm -I) Bull’s horn Human nail
Table 2 (continued)
W. Akhtar, H.G.M.
85
Edwards 1 Spectrochimica Acta Part A 53 (1997) 81-90
2.2. Raman spectroscopy
Fourier-transform Raman spectra were obtained using a Bruker IFS66 instrument with FRA106 Raman module attachment. 1064 nm wavelength excitation was effected using a Nd3 + / YAG laser with a maximum power of 200 mW and spectra were recorded using 4 cm - ’ spectral resolution. Generally, although the stronger features in the Raman spectra of keratotic tissue could be obtained with about 200 scans accumulation, spectral data were accumulated over 4000 scans to facilitate the observation of the weaker features in the spectra, in particular, the weaker bands in the region 1400-l 100 cm ~ ’ and less than 1000 cm - ’ which are critical for conformational studies.
3. Results and discussion Although the tissue samples studied here are all derived from keratin, there are some differences in the amino acid residue composition (Table 1) which may reflect the use to which the keratinized tissue is put. For example, of the four keratotic tissues for which analytical information is available in Table 1, the cystine content changes by 250% from human stratum corneum to bird’s feather quill; similarly, significant differences in the content of other amino acids between these materials can be noted such as glutamic acid, lysine, glycine and proline. In addition, animal claws, like teeth, contain crystalline hydroxyapatite and cystine-rich keratin, which confer on them their characteristic hardness. Unlike teeth, however, the hydroxyapatite in claws is not the major component but it does contribute to the survival of the proteinaceous material in buried or fossilized remains and is the reason for the survival of teeth, claws and bones when hard keratotic tissues have been biodeteriorated. Primate mammalian nails, although rich in cystine (Table 1) are not so calcified as animal claws; they are, therefore, classified as softer tissues. The wavenumbers and approximate assignments of the vibrational modes for the Raman spectra obtained in this study are shown in Table
Fig. 1. FT-Raman spectra of mammalian and avian keratotic samples;1064 nm excitation, 4000 scans, 4 cn- ’ spectral resolution, 200 mW power; wavenumber region, 400P 1800 cm-‘. (a) Bull’s horn (b) human nail (c) cat’s fur (d) cat’s claw.
2. Stack-plots of the Raman spectra are provided in Figs. 1-4, representing the v(CH) region, 2700-3200 cm-’ region, and the 1800-400 cm-’ region which contains the amide I and III features and information about the skeletal backbone and sulphur-sulphur bridging modes. 3.1. v(CH) region, 2700-3100
cm - l
All the samples studied in this work showed similar vibrational modes in the 2700- 3 100 cm ~ ’ region. Two weak features were reported in the 2719-2733 and 3060 cm-’ regions, and were
n
t
Fig. 2. FT-Raman spectra of mammalian and avian keratotic samples; conditions as for Fig. 1. (a) Callus tissue (b) feather quill (c) pheasant’s beak.
86
W. Akhtar, H.G.M. Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
Fig. 3. FT-Raman spectra of mammalian and avian keratotic samples; 1064 nm excitation, 4000 scans, 4 cm- ’ spectral resolution, 200 mW power;wavenumber region, 2500-3400 cm-‘. (a) Bull’s horn (b) human nail (c) cat’s fur (d) cat’s claw.
assigned to saturated aliphatic and olefinic C-H stretching vibrations, respectively. Two medium-strong intensity bands were also observed at approximately 2854 and 2871 cm-’ and were attributed to methylene C-H symmetric and methylene C-H asymmetric stretching, respectively. However, it should be noted that the asymmetric CH, vibrational band appeared at a slightly higher value for the feather quill sample, that is, 2880 cm - ‘. This band has also been reported in an earlier study of human stratum corneum [4,5], where a value of 2883 cm-’ was obtained and which was assigned to an asymmetrical C-H stretching vibration of methylene
(CH,) groups in lipid chains. A similar result has also been reported by Spiker and Levin [ 131, on their study of octacosane, palmitic acid, lysolecithin and DPPC. They observed strong features near 2850 and 2885 cm-’ in the C-H region, and compared the spectra with that of polycrystalline polyethylene in which transitions at 2848 and 2883 cm-’ were attributed to the symmetric and asymmetric CH, stretching modes. The most intense of the bands in the 2700-3100 cm-l region was assigned to the symmetrical CH, stretching vibration and was observed at around 2933 cm - ’ for all the keratin samples. Verma and Wallach [14] indicated that this band could have some structural implications; they observed that the unfolding of RNase produced a large increase in the Raman intensity at 2930 cm- ‘, which was interpreted as the exposure of previously buried aliphatic amino acid residues to the surrounding water. It has also been suggested that the 2933 cm-’ and 2871 cm ~ ’ bands form a Fermi resonance doublet through the interaction of the overtone of the CH, asymmetric deformation mode with the CH, symmetric stretching mode for the methyl groups at the acyl chain termini. The 2871 cm - l feature is more pronounced in infrared spectra, while the 2933 cm - ’ mode is more easily determined in the Raman spectrum of a lipid component. A band of medium intensity, which was assigned to the asymmetric stretch of the methyl (CH,) group, was also reported at approximately 2955 cm - ‘. Vibrational transitions for the C-H stretching modes have been assigned [13] for dipalmitoyl phosphatidylcholine (DPPC) and other structurally related molecules and it was concluded that, even though the assignments were made for phospholipid systems, it is likely that they are also relevant to other systems. The assignments for the modes in the 27003100 cm - ’ region are in agreement with those already reported for human stratum corneum and other keratotic biopolymers [3-51. 3.2. I500- 1700 cm - ’ region
Fig. 4. FT-Raman spectra of mammalian and avian keratotic samples; conditions as for Fig. 3. (a) Callus tissue (b) feather quill (c) pheasant’s beak.
This region contains valuable information on the amide I and II modes, v(CONH) and J(NH,),
W. Akhtar, H.G.M.
Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
ofthe keratins. A strong band was reported in the 1651-1657 cn-’ region for the mammalian samples and was attributed to the C=O stretching vibration of the amide I band, indicating that the mammalian keratins existed predominantly in the a-helix conformation [ 161. However, a shoulder was seen in the bull-horn and cat’s fur spectra at 1670 and 1668 cm - ’ respectively, indicating the presence of /I-keratin conformations [3]. The avian keratins also showed a band of strong intensity, but in the 1666 cm - ’ region, corresponding to the presence of the p-sheet structure as the dominant conformation. Other important modes included the amide II band which occurs in the 1550-1561 cm- ’ region, owing primarily to N-H in-plane bending with contributions from C-N stretching vibrations, and a C = C olefinic, stretching mode which appeared as a band of medium intensity in the 1616 cm-’ region. It is possible to assign other bands in this wavenumber region to tyrosine and phenylalanine vibrations, particularly those near 1605- 1613 cm-’ for the former [17] and 1585, 1605 cm-’ for the latter [18], which correspond to vg ring modes. The 1500- 1510 cm ~ ’ feature has been assigned in the literature to tyrosine [17].
3.3. 1500- 1200 cm ~ ’ region The main features of the 1.200- 1500 cm - ’ region are the C-H deformation and the amide III bands. All the samples studied showed weak bands in the 1241- 125 1 cm - ’ region, which indicated the presence of the amide III band, primarily a C-N stretch, with contributions from N-H in-plane bend and CH,-C stretching [19]. Random coil and a-sheet protein conformations show an amide III band in the 1240-1250 cm-’ region. The strongest band in this region is at 1449 cm-’ and is attributed to a methylene (CH,) deformation band (scissoring). Other methylene deformation bands were observed at 1298 and 1316 cm-‘. Methyl (CH,) deformation bands were also observed at 1414 cn-’ for all the keratotic samples.
87
3.4. v(CC) skeletal region; 1250- 1000 cm - ’ In this region we would expect v(CC) and v(CN) bands from the keratotic and lipoidal components of the tissues. Naturally, the features will be complex, but some assignments can be made on the basis of previous work on delipidised tissue ]81. From model phospholipid systems [15,20], it is also possible to assign C-C stretching modes for the skeletal region. The 1050- 1150 cm - ’ skeletal C-C stretching mode region in the Raman spectra directly reflects the intramolecular trans/ conformational changes within the gauche hydrophobic acyl chain matrix [21] of the bilayers. In the current work, the bands at approximately 1126, 1095-1107, and 1062 cm-’ were assigned to the three all-trans C-C stretching modes, although some of these bands, especially in the 1095-l 107 cm-’ region, are very weak. Wavenumbers of the 1127 cm ~ ’ band, attributed to an in-phase C-C motion, the weak intensity 1095-l 107 cm-’ region are functions of both chain length and temperature. Additionally, a weak band was observed in the 1080-1088 cm-’ region and was assigned to a random liquid-like conformation. Two bands, which were assigned to the cis conformation of the CCC backbone, were also reported in the 1030-1049 cm-’ region. Other C-C stretching vibrations included bands at 1208, 1174 and 1156 cm - ‘, all resulting from side chain amino acids [17,18]. The strongest intensity band in this region was observed at 1003 cm-’ for all the samples and is assigned to the C-C stretching vibration of the aromatic ring in the phenylalanine side chain. Lord and Yu [18] reported the same band at 1006 cm - ’ in RNase and concluded that it was owing to the breathing vibration of the monosubstituted ring in phenylalanine. 3.5. 500- 1000 cm - ’ region The most important spectral features in the 500-1000 cm-’ region are those of the sulphursulphur bond in the keratins. The relative intensities of the C-S and S-S stretching modes give a good indication of the relative sulphur content
W. Akhtar, H.G.M.
88 Table 3 v(SS) wavenumbers cystine residues Residue
Group
CYs
s-s
c-s
and
conformational
Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
dependence
Wavenumber @n-l)
Assignment
540
trans-gauche-trans gauche-gauche-tram gauche-gauche-gauche tram gauche
525 510 745-700 670-630
for
and the structural conformation of the disulphide linkages. In protein spectra the C-S vibrational band originates from methionine, cysteine and cystine. Most C-S stretching modes in the spectra of alkythiols appear in the 620-730 cm-’ region. For instance, CH,SH shows C-S stretching vibrations at 704 cm _ ‘, CH,CH,SH at 665 and 658 cm - ‘, and CH,CH(SH)CH, at 623 and 616 cm-‘. The wavenumber of the C-S stretching vibration is dependent on conformation in the environment of the C-S band. For example, for the truns form of methionine the C-S stretching vibrational bands appear at 665 and 724 cm-‘. In the gauche form [18] the C-S vibration gives a band at 700 cm - ‘. In the present study, for the mammalian and avian samples two weak bands were reported at around 643 and 621 cm-’ for all the samples and were assigned to the stretching vibrations of C-S bonds. Raman spectroscopy is a useful technique for the study of disulphide bonds owing to the S-S stretching vibrations in the 500-550 cm-’ region (Table 3). In proteins, the disulphide bond is part of a cystine residue or can be considered as two cysteine residues fused together through the oxidation of two sulphydryl groups. Among protein side-chain interactions, the disulphide bond is particularly important because it gives additional stability to the folding of a protein. In this study, all the samples analysed gave a band in the 510 cm-’ region for the S-S stretching mode, except for the human callus tissue sample. The 510 cm-’ value for the S-S
stretching vibration waveband is most commonly found for natural proteins, reflecting that the gauche-gauche-gauche conformation of CC-S-S-C-C (Fig. 5) is the most stable form, and indicating that naturally occurring proteins and peptides prefer to take the lowest energy conformation of disulphide bonds [22]. The only exception was the callus tissue samples, which gave a value of 529 cm - ‘, corresponding to the gauche-gauche-tram conformation (Fig. 5). A very important feature which was noticed in the callus spectra was the weak intensity of the S-S bond. Callus and human stratum corneum, are termed soft keratins, i.e. they do not contain much of the sulphur-containing amino acid cysteine, present as cysteic acid. The amount of cysteine in the callus samples is approximately 0.5% of the total amino acid content 1231 and this is evident from the weak intensity of the S-S stretching vibration; compared with the rest of the mammalian and avian samples where the amount of cysteine present can be between 6-10% of the total amino acid content [24], this being reflected in the stronger intensities as compared with the callus spectra. Two weak bands are found near 850 and 830 cm-l and are assigned to tyrosine side-chain vibrations involving CCH aromatic and aliphatic deformations, respectively [I 71. Of the Ramanactive tyrosine bands, the 850 and 830 cm-’ bands are considered to be useful for determining the environment of the tyrosine side chain [25]. The 850/830 cm-’ doublet is owing to Fermi resonance between the ring-breathing vibration and an overtone of an out-of-plane ring-breathing vibration of the puru-substituted benzene [26]. The relative intensity ratio of the doublet bands depends on the siting of the tyrosine in the skeletal framework of the protein [27]. In all the samples studied here, the 850 cm-’ band was of greater intensity than the 830 cm- ’ band, giving an Is5,,/Is3,, which is indicative of an exposed tyrosine ,side chain. This doublet has also been reported for tyrosine residues in ribonuclease A [28] and for neurotoxins isolated from the venom of sea snakes v91.
W. Akhtar, H.G.M.
Edwards 1 Spectrochimica Acta Part A 53 (1997) 81-90
Other features in this region include methylene (CH,) rocking modes near 885 cm-’ and CH, in-phase rocking modes near 740 and 755 cm-‘.
89
Lord and Yu [18] have reported similar bands at 761 and 879 cm - ’ and assigned these as indolering vibrations from tryptophan residues. Frushour and Koenig [30] have also reported that, besides the amide I and III bands, a band in the 890-945 cm-’ region is characteristic of an or-helical keratin conformation. This band arises from the skeletal C-C stretching vibration plus contributions from methyl rocking modes. This band has been reported at 907 cm ~’ for poly (L-Met) and 945 cm - ’ for poly (L-Lys), but is generally considered to be less reliable than the amide I and III bands for protein skeletal assignment [31].
4. Conclusion The results of this work have shown that it is possible to obtain good quality Raman spectra non-destructively from several mammalian and avian keratotic tissues with no sample preparation or pretreatment. From the molecular assignments proposed, it is possible to identify bands which are sensitive to skeletal backbone configuration of the keratotic protein. Examination of the v(SS) and v(CS) stretching region gives information about the local tram and gauche conformations about the sulphur-sulphur bonds for the cystine residues. The application of this technique to biomedical diagnosis involving keratinization of tissues is now open to study.
lb)
Cl.
2
Q”i’; CP.
a
CP
S,
Cc) Fig. 5. Diagramatic representation of CC-S-S-CC bond conformations of the disulphide bond in keratins (a) gauchegauche-gauche (b) gauche-gauche-tram (c) trans-gauche-
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PI H.G.M. Edwards, A.C. Williams and B.W. Barry, J. Mol. Structure, 347 (1995) 379. PI S. Keller, B. Schrader, A. Hoffman, W. Schrader, K. Metz, A. Rehlaender, J. Pahnke, M. Ruew and W. Budach, J. Raman Spectosc., 25 (1994) 663. [lOI H.P. Baden and L. Bonar, J. Invest. Derm., 51 (1968) 478. 1111W.G. Crewther, J.M. Gillespie, B.S. Harrap and A.S. Inglis, Biopolymers, 4 (1966) 905. WI B.S. Harrap and E.F. Woods, Biochem. J., 92 (1964) 19. R.C. Spiker and I.W. Levin, Biochim. Biophys. Acta, 388 1131 (1975) 361. v41 S.P. Verma and D.F.H. Wallach, Biochem. Biophys. Res. Commun., 74 (1977) 473. v51 I.W. Levin, in Advances in Infrared and Raman Spectroscopy, Vol. 11, R.J.H. Clark and R.E. Hester (Eds.). Wiley, Chichester, 1984. [I61 P.R. Carey, Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York, 1982. 1171 M. Pezolet, M. Pigeon-Gosselin and L. Coulombre, Biochim. Biophys. Acta, 453 (1976) 502. 1181 R.C. Lord and N.T. Yu, J. Mol. Biol., 50 (1970) 509; 51 (1970) 203. [19] T. Miyazawa, T. Shimanouchi and S. Mizushirna, J. Chem. Phys., 29 (1958) 611.
[20] J.L. Lippert and W.L. Peticolas, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 1572. [21] N. Yellin and I.W. Levin, Biochim. Biophys. Acta, 489 (1977) 177. [22] H. Sugeta, A. Go and T. Miyazawa, Chem. Letts., (1972) 83. [23] E.J. Wood and P.T. Bladon, The Human Skin, Studies in Biology Series, No. 164. E. Arnold, London, 1985. [24] R.D.B. Fraser, T.P. MacRae and G.E. Rogers, Keratins: Their Composition, Structure and Biosynthesis, C.C. Thomas, IL, USA, 1972. [25] N.T. Yu, B.H. Jo and D.C. O’Shea, Arch. Biochem. Biophys., 156 (1973) 71. [26] M.N. Siamwiza, R.C. Lord, M.C. Chen, T. Takamatsu, I Harada, H. Matsuura and T. Shimanouchi, Biochem., 14 (1975) 4870. [27] A.T. Tu, Raman Spectroscopy in Biology: Principles and Applications, Wiley, Chichester, 1982. [28] M.C. Chen and R.S. Lord, Biochem., 15 (1976) 1889. [29] N.T. Yu, T.S. Lui and A.T. Tu, J. Biol. Chem., 250 (1975) 1782. [30] B.G. Frushour and J.L. Koenig, Biopolymers, 14 (1975) 379. [31] G.D. Fasman, K. Itoh, C.S. Liu and R.C. Lord, Biopolymers, 17 (1978) 1729.
ELSEVIER
Spectrochimica
Fourier-transform
Acta Part A 53 (1997) 81-90
Raman spectroscopy of mammalian avian keratotic biopolymers W. Akhtar”,
Vhemistry
H.G.M.
and
Edwards”,*
and Chemical Technology, University of Bradford, Bradford BD7 IDP, UK
Received 27 March 1996; revised 23 May 1996; accepted 24 May 1996
Abstract The FT-Raman spectra of mammalian and avian keratotic biopolymers have been recorded, including bull’s horn, cat’s claw, bird’s feather quill, pheasant’s beak and compared with the hard keratinous tissue, human nail and callus. Although there were similarities in all the spectra, particularly in the v(CH) stretching region, the 1450-l 100 cm- 1 region exhibited some differences ascribed to intramolecular skeletal backbone conformational changes. Of particular significance for human, mammalian and avian samples in the 1000-400 cm _ ’ wavenumber region were differences in the structurally important v(SS) and v(CS) bands, near 500 cm-’ and 630 cm- I, respectively. The amide I and III modes near 1650 and 1250 cm- ’ respectively, demonstrate that the mammalian keratins studied exist predominantly in the a-helical conformation, whereas the avian keratins adopt the p-sheet structure as the dominant conformation. Keywords:
Avian keratotic biopolymers;
FT-Ramam
spectroscopy; Mammalian
1. Introduction Keratins are naturally occurring proteins which owe their rigidity and strength to sulphur-sulphur intramolecular cross-links between cysteine amino acid residues and to extensive intramolecular hydrogen bonding. Keratotic structural tissue is found in reptiles, birds and mammals and may also serve as a barrier to environmental stress. Keratins are the principal constituent of the outermost layer of human and animal skins and in appendages such as horns, hooves, hair and feath-
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biopolymers
ers. Whereas rhinoceros horn represents a primitive fibrous keratin, reptilian or squamate scales are highly sophisticated adaptations of different keratins in the same sample which fulfill specific purposes; the soft, flexible hinge of snake skin is mainly a-helical keratin whereas the hard scales are composed of layers of the p-sheet conformation. Some tissues have evolved from hair, e.g. animal horns, and their keratotic composition is quite similar. In some earlier studies from our laboratories, the keratotic tissue in the skins of reptiles and mammals, including three species of snake [1,2] and pig [3], were characterized using Raman spectroscopy and compared with human stratum
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W. Akhtar, H.G.M. Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
Table 1 Amino acid composition’ of mammalian and avian keratins Residue
Human stratum corneumb
Human nail”
Bird’s featheP
Bull’s horn’
alanine arginine aspartic acid half cystine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tyrosine valine
7.1 4.5 9.8 2.9 15.8 11.6 1.6 4.6 9.3 5.7 0.5 3.4 2.9 7.2 5.4 2.0 5.7
6.5 6.7 8.9 7.4 15.2 6.6 0.9 3.9 9.9 4.5 0.8 2.2 4.1 8.7 5.0 2.8 5.9
8.7 3.8 5.6 7.8 6.9 13.7 0.2 3.2 8.3 0.6 0.1 3.1 9.8 14.1 4.1 1.4 7.8
8.1 8.7 8.7 4.2 15.1 7.1 0.8 4.5 10.7 4.0 0.6 2.6 3.4 7.9 4.8 2.7 6.0
a Measured as residues per 100 amino acids. b Ref. [lo]. ’ Ref. [ll]. d Ref. [12].
corneum [4,5], the hard outer layer of the epidermis which consists of keratin cells in a lipoidal matrix. Currently, knowledge of the keratotic structures of tissues is providing some useful information for the pharmaceutical therapeutic control of skin disease in humans and is essential for the understanding of transdermal drug delivery systems [6] and their operation in, for example, hormone replacement therapy using patches applied to the stratum corneum [7]. In this context, it has been shown that reptilian squamate keratins are not good models for human stratum corneum, despite their adoption for this purpose for drug diffusion studies [3]. Also, in the area of biomedical diagnostics, the application of Raman spectroscopic techniques [8,9] to the study of healthy and diseased skin tissue is providing novel information about the origins of skin afflictions and their treatment, e.g. psoriasis, skin cancer, melanomas and sunburn. When it is realised that almost 40% of all accidents in the workplace are skin-related and up to 10% of the population are affected by skin disorders such as acne, vitiligo, psoriasis and cancer, the importance of an understanding of keratin composition and
the spectroscopic changes therein for interpretation of Raman spectra on a diagnostic basis is of great significance. In this study, we report the Raman spectroscopic analysis of keratins from mammalian and avian tissues, including human samples, from which novel information is provided about molecular vibrational assignments and keratotic structure.
2. Experimental 2.1. Samples
In this work, representative samples of different keratotic tissues from mammals and birds were taken. A bull’s horn (Texas longhorn), cat’s claw (domestic cat) and pheasant’s beak represent mammalian and avian hard tissue; bird’s feather (pigeon) quill and cat’s fur represented softer tissue, and human nail and callus skin tissue samples were analysed to provide a comparison with the hard and soft keratotic samples from birds and mammals.
1448 1416
1338 1316 1266 1250 1207 1176 1156 1126 1096 1086 1061 1043 1030 1003 956 936
1385 1340 1318
1271 1251 1207 1174 1154 1126 1098 1084 1066 1051 1031 1003 961 934
1386 1341 1316 1296 1269 1249 1207 1175 1156 1126 1106 1088 1063 1048 1031 1003 956 937
1519
1585 1556
1657 1615
1449 1416
1652 1618 1608 1585 1552
1449 1422 -
1554 -
1670 1653 1615 -
2957 2931 2871 2855 2733
2955 2931 2871 2859 2731
Cat’s claw
1269 1245 1207 1175 1156 1127 1098 1086 1062 1043 1036 1003 958 938
1448 1416 1405 1385 1340 1315 1340 1318 1298 1269 1251 1208 1175 1157 1127 1107 1084 1062 1049 1032 1003 957 937
1449 1421
1651 1618 1606 1581 1554
1270 1241 1210 1174 1154 1126 1095 1085 1061 1045 1030 1003 956 935
1395 1342 1317
,^, - - _
1269 1243 1208 1174 1158 1126 1095 1080 1060 1049 1031 1003 959 937
1390 1340 1314
1449 1414
1523
1524 1508 1500 1449 1414
1667
2957 2935 2875 2853 2730
3064
Pheasant’s
of mammalian
1615 1606 1587 1556 -
quill
modes
1615 1608 1585 1550
1665
2960 2933 2880 2856 2726
2958 2933 2871 2856 2719
2953 2933 2874 2855
1668 1654 1616 1610 1587 1559 1533 1527 1519
3060
Feather
3061
tissue
of the vibrational
3060
Callus
descriptions
Cat’s fur
and approximate
3062
nail
(cm-‘)
3062
(cm-‘) Human
Wavenumber Bull’s horn
3062 3002 2955 2930 2873 2857 2730
wavenumbers
Table 2 FT-Raman
,_1
-
beak
_
keratotic
biopolymers
band
/_
,”
_1
R.
_.
.,,
,_,
in
,_
_,
,
_
v(CC) v(CC) v(CC); 6 (COH) v(CC) skeletal, tram conformation v(CC) skeletal, tram conformation v(CC) skeletal, random conformation v(CC) skeletal, tram conformation v(CC) skeletal, cis conformation v(CC) skeletal, cis conformation v(CC) aromatic ring p (CH,); b (CCH) olefinic p(CH,) terminal; v(CC) a-helix
I
6(CH) 6 (CHJ 6 (CH,) r(CN); s(NH). Amide III. cc-helix 6(CH,) wagging; v(CN). Amide III. Disordered
6 (CH,) 6[C(CH,),] symmetric 6 (CH,) symmetric
@NH) v(C = C) carotenoid v(C = C) carotenoid v(C = C) carotenoid 6(CH,) scissoring
v(CH) olefinic r(CH) aromatic v(CH,) asymmetric v(CH,) symmetric v(CHJ asymmetric r(CH,) symmetric “(CH) aliphatic (overtone and combination of 6(CH,) fundamentals) v(C = 0) Amide I p-sheet v(C = 0) Amide I a-helix v(C = C) olefinic v(C = C) olefinic v(C = C) olefinic s(NH), r(CN). Amide II
Assignment
and avian
8
494
509
146 644 622 603
852 829
411
v(SS) [gauche-gauche-gauche] v(SS) [gauche-gauche-gauche] 6 (CCC) skeletal
492 417
512
512
509
529 508
510
136 642 621 603
P (CM 6 (CCH) aromatic 6 (CCH) aliphatic p (CH,) in-phase p (CH,) in-phase V(G) [gauche] v(CS) [gauche] P (CH) wagging v(SS) [gaucheegauche-tram] 882 853 828 151 740 643 622
880 853 828 156 742 643 621
885 853 830 758 744 644 622
893 854 829
892 852 829 756 146 643 621
896 852 829 157 743 643 621
Assignment Pheasant’s beak
Feather quill
Callus tissue
Cat’s fur
Cat’s claw
Wavenumber (cm -I) Bull’s horn Human nail
Table 2 (continued)
W. Akhtar, H.G.M.
85
Edwards 1 Spectrochimica Acta Part A 53 (1997) 81-90
2.2. Raman spectroscopy
Fourier-transform Raman spectra were obtained using a Bruker IFS66 instrument with FRA106 Raman module attachment. 1064 nm wavelength excitation was effected using a Nd3 + / YAG laser with a maximum power of 200 mW and spectra were recorded using 4 cm - ’ spectral resolution. Generally, although the stronger features in the Raman spectra of keratotic tissue could be obtained with about 200 scans accumulation, spectral data were accumulated over 4000 scans to facilitate the observation of the weaker features in the spectra, in particular, the weaker bands in the region 1400-l 100 cm ~ ’ and less than 1000 cm - ’ which are critical for conformational studies.
3. Results and discussion Although the tissue samples studied here are all derived from keratin, there are some differences in the amino acid residue composition (Table 1) which may reflect the use to which the keratinized tissue is put. For example, of the four keratotic tissues for which analytical information is available in Table 1, the cystine content changes by 250% from human stratum corneum to bird’s feather quill; similarly, significant differences in the content of other amino acids between these materials can be noted such as glutamic acid, lysine, glycine and proline. In addition, animal claws, like teeth, contain crystalline hydroxyapatite and cystine-rich keratin, which confer on them their characteristic hardness. Unlike teeth, however, the hydroxyapatite in claws is not the major component but it does contribute to the survival of the proteinaceous material in buried or fossilized remains and is the reason for the survival of teeth, claws and bones when hard keratotic tissues have been biodeteriorated. Primate mammalian nails, although rich in cystine (Table 1) are not so calcified as animal claws; they are, therefore, classified as softer tissues. The wavenumbers and approximate assignments of the vibrational modes for the Raman spectra obtained in this study are shown in Table
Fig. 1. FT-Raman spectra of mammalian and avian keratotic samples;1064 nm excitation, 4000 scans, 4 cn- ’ spectral resolution, 200 mW power; wavenumber region, 400P 1800 cm-‘. (a) Bull’s horn (b) human nail (c) cat’s fur (d) cat’s claw.
2. Stack-plots of the Raman spectra are provided in Figs. 1-4, representing the v(CH) region, 2700-3200 cm-’ region, and the 1800-400 cm-’ region which contains the amide I and III features and information about the skeletal backbone and sulphur-sulphur bridging modes. 3.1. v(CH) region, 2700-3100
cm - l
All the samples studied in this work showed similar vibrational modes in the 2700- 3 100 cm ~ ’ region. Two weak features were reported in the 2719-2733 and 3060 cm-’ regions, and were
n
t
Fig. 2. FT-Raman spectra of mammalian and avian keratotic samples; conditions as for Fig. 1. (a) Callus tissue (b) feather quill (c) pheasant’s beak.
86
W. Akhtar, H.G.M. Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
Fig. 3. FT-Raman spectra of mammalian and avian keratotic samples; 1064 nm excitation, 4000 scans, 4 cm- ’ spectral resolution, 200 mW power;wavenumber region, 2500-3400 cm-‘. (a) Bull’s horn (b) human nail (c) cat’s fur (d) cat’s claw.
assigned to saturated aliphatic and olefinic C-H stretching vibrations, respectively. Two medium-strong intensity bands were also observed at approximately 2854 and 2871 cm-’ and were attributed to methylene C-H symmetric and methylene C-H asymmetric stretching, respectively. However, it should be noted that the asymmetric CH, vibrational band appeared at a slightly higher value for the feather quill sample, that is, 2880 cm - ‘. This band has also been reported in an earlier study of human stratum corneum [4,5], where a value of 2883 cm-’ was obtained and which was assigned to an asymmetrical C-H stretching vibration of methylene
(CH,) groups in lipid chains. A similar result has also been reported by Spiker and Levin [ 131, on their study of octacosane, palmitic acid, lysolecithin and DPPC. They observed strong features near 2850 and 2885 cm-’ in the C-H region, and compared the spectra with that of polycrystalline polyethylene in which transitions at 2848 and 2883 cm-’ were attributed to the symmetric and asymmetric CH, stretching modes. The most intense of the bands in the 2700-3100 cm-l region was assigned to the symmetrical CH, stretching vibration and was observed at around 2933 cm - ’ for all the keratin samples. Verma and Wallach [14] indicated that this band could have some structural implications; they observed that the unfolding of RNase produced a large increase in the Raman intensity at 2930 cm- ‘, which was interpreted as the exposure of previously buried aliphatic amino acid residues to the surrounding water. It has also been suggested that the 2933 cm-’ and 2871 cm ~ ’ bands form a Fermi resonance doublet through the interaction of the overtone of the CH, asymmetric deformation mode with the CH, symmetric stretching mode for the methyl groups at the acyl chain termini. The 2871 cm - l feature is more pronounced in infrared spectra, while the 2933 cm - ’ mode is more easily determined in the Raman spectrum of a lipid component. A band of medium intensity, which was assigned to the asymmetric stretch of the methyl (CH,) group, was also reported at approximately 2955 cm - ‘. Vibrational transitions for the C-H stretching modes have been assigned [13] for dipalmitoyl phosphatidylcholine (DPPC) and other structurally related molecules and it was concluded that, even though the assignments were made for phospholipid systems, it is likely that they are also relevant to other systems. The assignments for the modes in the 27003100 cm - ’ region are in agreement with those already reported for human stratum corneum and other keratotic biopolymers [3-51. 3.2. I500- 1700 cm - ’ region
Fig. 4. FT-Raman spectra of mammalian and avian keratotic samples; conditions as for Fig. 3. (a) Callus tissue (b) feather quill (c) pheasant’s beak.
This region contains valuable information on the amide I and II modes, v(CONH) and J(NH,),
W. Akhtar, H.G.M.
Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
ofthe keratins. A strong band was reported in the 1651-1657 cn-’ region for the mammalian samples and was attributed to the C=O stretching vibration of the amide I band, indicating that the mammalian keratins existed predominantly in the a-helix conformation [ 161. However, a shoulder was seen in the bull-horn and cat’s fur spectra at 1670 and 1668 cm - ’ respectively, indicating the presence of /I-keratin conformations [3]. The avian keratins also showed a band of strong intensity, but in the 1666 cm - ’ region, corresponding to the presence of the p-sheet structure as the dominant conformation. Other important modes included the amide II band which occurs in the 1550-1561 cm- ’ region, owing primarily to N-H in-plane bending with contributions from C-N stretching vibrations, and a C = C olefinic, stretching mode which appeared as a band of medium intensity in the 1616 cm-’ region. It is possible to assign other bands in this wavenumber region to tyrosine and phenylalanine vibrations, particularly those near 1605- 1613 cm-’ for the former [17] and 1585, 1605 cm-’ for the latter [18], which correspond to vg ring modes. The 1500- 1510 cm ~ ’ feature has been assigned in the literature to tyrosine [17].
3.3. 1500- 1200 cm ~ ’ region The main features of the 1.200- 1500 cm - ’ region are the C-H deformation and the amide III bands. All the samples studied showed weak bands in the 1241- 125 1 cm - ’ region, which indicated the presence of the amide III band, primarily a C-N stretch, with contributions from N-H in-plane bend and CH,-C stretching [19]. Random coil and a-sheet protein conformations show an amide III band in the 1240-1250 cm-’ region. The strongest band in this region is at 1449 cm-’ and is attributed to a methylene (CH,) deformation band (scissoring). Other methylene deformation bands were observed at 1298 and 1316 cm-‘. Methyl (CH,) deformation bands were also observed at 1414 cn-’ for all the keratotic samples.
87
3.4. v(CC) skeletal region; 1250- 1000 cm - ’ In this region we would expect v(CC) and v(CN) bands from the keratotic and lipoidal components of the tissues. Naturally, the features will be complex, but some assignments can be made on the basis of previous work on delipidised tissue ]81. From model phospholipid systems [15,20], it is also possible to assign C-C stretching modes for the skeletal region. The 1050- 1150 cm - ’ skeletal C-C stretching mode region in the Raman spectra directly reflects the intramolecular trans/ conformational changes within the gauche hydrophobic acyl chain matrix [21] of the bilayers. In the current work, the bands at approximately 1126, 1095-1107, and 1062 cm-’ were assigned to the three all-trans C-C stretching modes, although some of these bands, especially in the 1095-l 107 cm-’ region, are very weak. Wavenumbers of the 1127 cm ~ ’ band, attributed to an in-phase C-C motion, the weak intensity 1095-l 107 cm-’ region are functions of both chain length and temperature. Additionally, a weak band was observed in the 1080-1088 cm-’ region and was assigned to a random liquid-like conformation. Two bands, which were assigned to the cis conformation of the CCC backbone, were also reported in the 1030-1049 cm-’ region. Other C-C stretching vibrations included bands at 1208, 1174 and 1156 cm - ‘, all resulting from side chain amino acids [17,18]. The strongest intensity band in this region was observed at 1003 cm-’ for all the samples and is assigned to the C-C stretching vibration of the aromatic ring in the phenylalanine side chain. Lord and Yu [18] reported the same band at 1006 cm - ’ in RNase and concluded that it was owing to the breathing vibration of the monosubstituted ring in phenylalanine. 3.5. 500- 1000 cm - ’ region The most important spectral features in the 500-1000 cm-’ region are those of the sulphursulphur bond in the keratins. The relative intensities of the C-S and S-S stretching modes give a good indication of the relative sulphur content
W. Akhtar, H.G.M.
88 Table 3 v(SS) wavenumbers cystine residues Residue
Group
CYs
s-s
c-s
and
conformational
Edwards / Spectrochimica Acta Part A 53 (1997) 81-90
dependence
Wavenumber @n-l)
Assignment
540
trans-gauche-trans gauche-gauche-tram gauche-gauche-gauche tram gauche
525 510 745-700 670-630
for
and the structural conformation of the disulphide linkages. In protein spectra the C-S vibrational band originates from methionine, cysteine and cystine. Most C-S stretching modes in the spectra of alkythiols appear in the 620-730 cm-’ region. For instance, CH,SH shows C-S stretching vibrations at 704 cm _ ‘, CH,CH,SH at 665 and 658 cm - ‘, and CH,CH(SH)CH, at 623 and 616 cm-‘. The wavenumber of the C-S stretching vibration is dependent on conformation in the environment of the C-S band. For example, for the truns form of methionine the C-S stretching vibrational bands appear at 665 and 724 cm-‘. In the gauche form [18] the C-S vibration gives a band at 700 cm - ‘. In the present study, for the mammalian and avian samples two weak bands were reported at around 643 and 621 cm-’ for all the samples and were assigned to the stretching vibrations of C-S bonds. Raman spectroscopy is a useful technique for the study of disulphide bonds owing to the S-S stretching vibrations in the 500-550 cm-’ region (Table 3). In proteins, the disulphide bond is part of a cystine residue or can be considered as two cysteine residues fused together through the oxidation of two sulphydryl groups. Among protein side-chain interactions, the disulphide bond is particularly important because it gives additional stability to the folding of a protein. In this study, all the samples analysed gave a band in the 510 cm-’ region for the S-S stretching mode, except for the human callus tissue sample. The 510 cm-’ value for the S-S
stretching vibration waveband is most commonly found for natural proteins, reflecting that the gauche-gauche-gauche conformation of CC-S-S-C-C (Fig. 5) is the most stable form, and indicating that naturally occurring proteins and peptides prefer to take the lowest energy conformation of disulphide bonds [22]. The only exception was the callus tissue samples, which gave a value of 529 cm - ‘, corresponding to the gauche-gauche-tram conformation (Fig. 5). A very important feature which was noticed in the callus spectra was the weak intensity of the S-S bond. Callus and human stratum corneum, are termed soft keratins, i.e. they do not contain much of the sulphur-containing amino acid cysteine, present as cysteic acid. The amount of cysteine in the callus samples is approximately 0.5% of the total amino acid content 1231 and this is evident from the weak intensity of the S-S stretching vibration; compared with the rest of the mammalian and avian samples where the amount of cysteine present can be between 6-10% of the total amino acid content [24], this being reflected in the stronger intensities as compared with the callus spectra. Two weak bands are found near 850 and 830 cm-l and are assigned to tyrosine side-chain vibrations involving CCH aromatic and aliphatic deformations, respectively [I 71. Of the Ramanactive tyrosine bands, the 850 and 830 cm-’ bands are considered to be useful for determining the environment of the tyrosine side chain [25]. The 850/830 cm-’ doublet is owing to Fermi resonance between the ring-breathing vibration and an overtone of an out-of-plane ring-breathing vibration of the puru-substituted benzene [26]. The relative intensity ratio of the doublet bands depends on the siting of the tyrosine in the skeletal framework of the protein [27]. In all the samples studied here, the 850 cm-’ band was of greater intensity than the 830 cm- ’ band, giving an Is5,,/Is3,, which is indicative of an exposed tyrosine ,side chain. This doublet has also been reported for tyrosine residues in ribonuclease A [28] and for neurotoxins isolated from the venom of sea snakes v91.
W. Akhtar, H.G.M.
Edwards 1 Spectrochimica Acta Part A 53 (1997) 81-90
Other features in this region include methylene (CH,) rocking modes near 885 cm-’ and CH, in-phase rocking modes near 740 and 755 cm-‘.
89
Lord and Yu [18] have reported similar bands at 761 and 879 cm - ’ and assigned these as indolering vibrations from tryptophan residues. Frushour and Koenig [30] have also reported that, besides the amide I and III bands, a band in the 890-945 cm-’ region is characteristic of an or-helical keratin conformation. This band arises from the skeletal C-C stretching vibration plus contributions from methyl rocking modes. This band has been reported at 907 cm ~’ for poly (L-Met) and 945 cm - ’ for poly (L-Lys), but is generally considered to be less reliable than the amide I and III bands for protein skeletal assignment [31].
4. Conclusion The results of this work have shown that it is possible to obtain good quality Raman spectra non-destructively from several mammalian and avian keratotic tissues with no sample preparation or pretreatment. From the molecular assignments proposed, it is possible to identify bands which are sensitive to skeletal backbone configuration of the keratotic protein. Examination of the v(SS) and v(CS) stretching region gives information about the local tram and gauche conformations about the sulphur-sulphur bonds for the cystine residues. The application of this technique to biomedical diagnosis involving keratinization of tissues is now open to study.
lb)
Cl.
2
Q”i’; CP.
a
CP
S,
Cc) Fig. 5. Diagramatic representation of CC-S-S-CC bond conformations of the disulphide bond in keratins (a) gauchegauche-gauche (b) gauche-gauche-tram (c) trans-gauche-
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[20] J.L. Lippert and W.L. Peticolas, Proc. Natl. Acad. Sci. U.S.A., 68 (1971) 1572. [21] N. Yellin and I.W. Levin, Biochim. Biophys. Acta, 489 (1977) 177. [22] H. Sugeta, A. Go and T. Miyazawa, Chem. Letts., (1972) 83. [23] E.J. Wood and P.T. Bladon, The Human Skin, Studies in Biology Series, No. 164. E. Arnold, London, 1985. [24] R.D.B. Fraser, T.P. MacRae and G.E. Rogers, Keratins: Their Composition, Structure and Biosynthesis, C.C. Thomas, IL, USA, 1972. [25] N.T. Yu, B.H. Jo and D.C. O’Shea, Arch. Biochem. Biophys., 156 (1973) 71. [26] M.N. Siamwiza, R.C. Lord, M.C. Chen, T. Takamatsu, I Harada, H. Matsuura and T. Shimanouchi, Biochem., 14 (1975) 4870. [27] A.T. Tu, Raman Spectroscopy in Biology: Principles and Applications, Wiley, Chichester, 1982. [28] M.C. Chen and R.S. Lord, Biochem., 15 (1976) 1889. [29] N.T. Yu, T.S. Lui and A.T. Tu, J. Biol. Chem., 250 (1975) 1782. [30] B.G. Frushour and J.L. Koenig, Biopolymers, 14 (1975) 379. [31] G.D. Fasman, K. Itoh, C.S. Liu and R.C. Lord, Biopolymers, 17 (1978) 1729.