Fourier transform Raman and IR spectra of snake skin

SpeetroehimicaActa. Vol. 49A, No. 516. pp. 801-807, 1993 Printed in Great Britain

0584-8539/93 $6.00 + 0.00 @_I1993 Pergamon Press Ud

Fourier transform Raman and IR spectra of snake skin B. W. BARRY and A. C. WILLIAMS Postgraduate Studies in Pharmaceutical Technology, School of Pharmacy, University of Bradford, Bradford, West Yorkshire, BD7 lDP, U.K.

and H. G. M. EDWARDS Chemistry and Chemical Technology, University of Bradford, Bradford, West Yorkshire, BD7 IDP, U.K. (Received 3 June 1992; accepted 26 August 1992) Abstract-The Fourier transform (PI’) Raman and IR spectra of the shed dorsal skin of the snake Elaphe obsolete (American black rat snake) are reported. Vibrational spectroscopic assignments are proposed for the first time. Although good quality Raman spectra were obtained from the hinge regions using an FT Raman microscope, the dorsal scale regions fluoresced even with 1064nm IR excitation. This was ascribed to pigmentation markings on the scales.

INTRODUCTION THE absorption

of materials through human skin is an important process which is currently receiving attention in biomedical science, particularly in the field of drug delivery. However, there are many problems associated with studies on human skin caused by difficulties in the supply and standardization of post mortem or surgical samples for percutaneous absorption experiments. For this reason in vitro animal skins have been considered for these studies, particularly in recent years those of snakes and the hairless mouse [ 1,2]. Biomedically and chemically the use of shed squamate epidermis as a model membrane for human skin has several advantages, one of the most important being a plentiful supply obtained by natural shedding from the animal without killing it [3]. Another useful feature of shed snake skin, unlike that removed surgically from human cadavers, is the absence of a biohazard rating with consequent containment and handling problems for the presentation of samples to spectrometers for analysis. Snake epidermis has a complex structure based on a hinge and scale construction [4]. It is believed [5] that the hinges consist primarily of flexible /?-keratin and that the scales contain keratin folds with entrapped pigmentation. Beneath the keratin layers are regions of lipid lamellae. Although there are reports in the literature of Raman spectroscopic studies of neurotoxins in sea-snake venom [6-81, there has been no previous spectroscopic study of snake skin. In this work we report the results of a Fourier transform (ET) Raman microscopic and ET-IR study of the shed skin of the American black rat snake, Eluphe obsokta, which has been used in permeability barrier tests for chemically enhanced drug delivery, as well as simple drug diffusion studies [l, 21.

EXPERIMENTAL

Skin samples Dry shed skin samples of Elaphe obsoleta were mounted on specially prepared microscope slides with 5 mm holes drilled through. This sampling arrangement proved to be necessary for the Raman microscopic studies because background glass reflections interfered with the very weak Raman spectra from the skin sections. The samples were taken from the dorsal region, consisting of relatively large, dark brown marked scales with paler hinges. The stripping technique using 801

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adhesive tape was also applied for the removal of surface layers and exposure of lipid layers underneath [ 11. Raman spectra

Raman spectra were excited at 1064nm using a YAG laser with a maximum power of 350 mW and recorded using a Bruker IFS 66 with FRA 106 IT-Raman attachment and coupled to a microscope which operated with a 40X objective. The samples were not prone to thermal degradation at the incident laser power levels used in the experiments but the spectra were very weak and hence required about 3000 scans at 8 cm-’ spectral resolution typically to achieve good signal-to-noise ratios. Normally, scan times were about 2 h with the germanium detector cooled to liquid nitrogen temperatures over the scan range, A0 = 100-3500 cm-‘. The sample spot size was 50 urn. Spectral response was corrected for white light and the observed band wavenumbers calibrated against the internal laser frequency were correct to better than f 1 cm-‘. Infrared spectra

FI-IR spectra in transmission were obtained over the wavenumber range 4000-400 cm-’ with a spectral resolution of 4 cm-’ using a Mattson Instruments 6020 Galaxy Series spectrometer with DTGS detector. RESULTS AND DISCUSSION

The snake skin surface is shown in Fig. 1; the scale construction of a dorsal sample under low magnification (A) exhibits finer structural detail with lOO-fold higher magnification of scale (B) and hinge (C) regions. The characteristic markings of the skin arise from pigmentation within the keratin folds of the scales [9, lo], whereas the hinges themselves are quite colourless. Raman spectra of the dorsal surface hinges were successfully obtained and an example is shown in Fig. 2, with an expanded region 2000-400 cm-’ in Fig. 3 to illustrate better the points made in the text. The FT-IR spectrum of the scale and hinge region is shown in Fig. 4. Raman spectra of the scale region could not be obtained with the same quality as that of the hinges because of sample fluorescence ascribed to the pigmentation held between keratin layers in the scales. The vibrational wavenumbers and approximate assignments of the vibrational modes are given in Table 1. Several points are immediately apparent from the spectra in Figs 2 and 3 and wavenumber assignments in Table 1, namely: (i) although the shed skin samples are relatively dry at ambient conditions, the presence of water trapped in the lipid and keratin layers swamps the IR spectral region around 2900-3500 cm-‘; (ii) the Raman and IR spectra exhibit many differences in band intensity and wavenumbers, with several coincidences; (iii) the IR spectrum below 1400 cm-’ is almost featureless and devoid of spectral information, unlike the Raman spectrum which provides some important information about functionality and skeletal conformation. The additional time involved in recording the Raman spectra compared with the IR must therefore be considered in terms of the type of information sought from the experiments. This is particularly important for our future studies which will involve the vibrational spectroscopic evaluation of systems for enhanced drug delivery through chemical modification of the skin surface [ll]. For example, molecular effects upon the Y(C=O) stretching and s(NH) bending wavenumbers of the amide linkages around 1500-1700 cm-’ will be best studied in the IR, where the spectral intensity is greatest. The v(CH) region which could be used to monitor the skin lipids and proteins is accessible by IR or Raman techniques but the presence of a variable content of water entrapped in the skin will tend to favour the Raman spectra here. However, the Raman experiments provide more information in the region below 14OOcm-‘, where the IR spectra are diffuse, perhaps because of the presence of skin water absorption.

FI’-Raman and IR spectra

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(A)

Fig. 1. Photographs of dorsal shed skin samples of Eraphe obsoleta showing the hinge and scale construction and markings on the scales: (A) general view, low magnification 10 x ; (B) scale, 1000 x ; (C) hinge, 1000 x .

We shall now consider the assignments (Table 1) of the observed Raman and IR spectra according to the following wavenumber regions: (a) 2700-31OOcm-‘: five medium or medium-strong intensity bands appear in the Raman and IR spectra of the snake skin hinge. There is generally a good correlation between the Raman and the IR spectra and the assignments follow from standard work on organic and biomolecules in the literature [12-151. The major differences between the IR and Raman spectra in this region occur around 2960 cm-‘, where the strong IR band assigned to v(CH,) stretching at 2962 cm-’ is observed as two medium intensity bands at 2956, 2975 cm-’ in the Raman spectrum. The medium intensity IR feature at 3075 cm-’ is probably the first overtone of the very strong Y(CO), amide II, band at 1543 cm-’ U(A)49:5/6-N

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(2 x 1543=3086cm-‘). Two weak features in the Raman at 2720 and 306Ocm-’ are assigned to olefinic and saturated aliphatic Y(CH) stretching, respectively. (b) 1500-1700 cm-‘: this wavenumber region contains the most intense features in the IR spectrum, arising from the -CONH- grouping, predominantly from protein structure, namely the amide I and amide II bands at 1657 and 1547 cm-‘, respectively. These bands are much weaker in the Raman spectrum at 1656 and 154Ocm-‘. Other important features in this region are the Y(C=C) olefinic stretching mode at 1614cm-’ in the Raman and several strong features in the IR around 1520-1560 cm-‘, which are assigned to s(NH) and Y(CO) modes typical of proteins. (c) 1200-1500 cm-‘: this region contains several bands of medium and strong intensity in the IR and rather weaker features in the Raman. Here, the bands have been assigned to 6(CHJ modes around 1350-1470 cm-’ and d(CH2) wagging modes around 1250 cm-‘. (d) 400-1200 cm-‘: a region of weak, diffuse bands in the IR ascribed to energy loss arising from entrapped water absorption in the samples. Although the observed bands are also quite weak in the Raman spectra, they have been observed reproducibly in several samples run under different conditions and are therefore assigned to real features. Some critically important skeletal molecular information is contained in this spectral region and bands attributable to cis-, fruns- and random conformations of the CCC backbone of lipids are identifiable. In the lower wavenumbers of this spectral region the &CH2) modes can be observed in the IR and Raman spectra and a broad feature at 491 cm-’ is assigned to the Y(SS) stretching mode of sulphur bridges in the keratin protein [16,17]. The stripped dorsal samples exhibit some intensity differences and several new features around 1300-1500 cm-’ which will be the subject of further study. It is believed that the stripping technique applied to the shed snake skin in the present work removed surface keratin layers and exposed fresh lipid biolayers beneath. The depth of penetration of the IR radiation used here will be of importance for the evaluation of the molecular changes on progression through the squamate epidermis. A comparison of the IR spectra of the dorsal and stripped dorsal skin samples is also given in Fig. 4. From this, new features in the stripped dorsal samples at 2930, 1450, 1431, 1399, 1342 and 972 cm-’ are identified and intensity changes of the bands near 2900,150O and 1300 cm-’ are noted. 1

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Wavenumber (cm-l) Fig. 2. FT-Raman microscope spectrum of dorsal shed snake skin hinge from Elaphe obsoleta, AB = 100-3500 cm-‘; 300 scans, 8 cm-’ spectral resolution, laser power 300 mW at 1064 nm; 40 X magnification.

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Raman shift (cm-‘) Fig. 3. Expanded region of Fig. 2, Ai, = 400-2000 cm-‘.

The problems associated with the study of proteins in biological materials by IR spectroscopy have been highlighted in recent work by MANSTCHet al. [18] and can be clearly identified in Fig. 4. Broad water absorptions make the identification of amide and skeletal structures difficult. Also, the assignment of new features to changes in protein structure is complicated by the presence of impurities with high molar absorptivities, E. Quantitative analysis is especially difficult and dependent on assumptions of similar E values for ordered and disordered structures and on the accurate deconvolution of weak features on the broad absorption background. Comparison and critical evaluation of the IR and Raman methods for skin samples is the subject of another paper [19]. This study represents the first reported vibrational spectroscopic analysis of shed squamate skin. The biomedical applications are important at the molecular level in the

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Wavenumber (cm-l) Fig. 4. FT-IR spectra of shed snake skin from Elaplte obsoleta, Y = 400-3750 cm-‘; dorsal and stripped dorsal regions; 64 scans at 4 cm-’ spectral resolution.

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Table 1. Vibrational wavenumbers (reciprocal centimetres) and assignments for the shed skin of the snake, Elaphe obsoleta: Raman assignments for dorsal hinge region; IR for bulk dorsal (hinge and scale) region

Infrared 32% vs. br 3075m

306ow 30@2w 2975 mw, sh 2962 s 2926 s 2875 m 2852 m

Approximate description of vibrational mode

Raman

2956 mw, sh 2933 s 2882 m 2852 mw, sh 2720 VW

v(OH) of Hz0 1st overtone, amide II at 1547 cm-’ v(CH) olefinic v(CH) aromatic v(CHS) asymmetric v(CH,) asymmetric v(CH,) asymmetric vCH,) asymmetric v(CH,) symmetric v(CH,) symmetric v(CH) aliphatic

1691 w, sh 1657 s 1649 s 1641 s 1632 vs

1672 mw, sh 1656 ms

1614 mw 1562 m, sh 1547 YS 1536 s, br 1517 m, sh 1501 w, sh 1469 s 1451 s, br 1439 m, sh

1540 mw

6(NH) 1510 mw 1473 w, sh 1452 m 1417 w

1405 m 1391 s 1369 m 1339 m 1313 w

1394 w

a(CHz) 6(CH2) asymmetric S(CHJ scissoring ~(CHI) a[C(CH,),] symmetric 6(CHg) symmetric d(C-CHg) symmetric

1351 mw, sh 1332 mw 1294 mw

1278 w, sh 1242 s

v(C=O) amide I b-sheet v(C=O) amide I a-helix v(C=O) amide 1 a-helix v(C=O) amide I v(C=O) amide I v(C=C) oletinic v(C=C) olefinic v(CN) and 6(NH) amide II

1267 w 1240 mw

v(CN) amide III C(CHJ twisting v(NH), v(CN) a-helix amide III 6(CHJ wagging 6(CH2) wagging; v(CN) amide III disordered

1217 w, sh 1205 w, sh 1174w 1159 w, sh, br 1128m 1103 m

v(CC) 1197 w, sh 1170w 1151 w 1124 mw 1085 w

1075 s

v(CC) v(CC); 6(COH) v(CC) skeletal tnw conformation v(CC) skeletal random conformation v(CC) skeletal tranr conformation

1055 VW 104.5w. sh

931 w, br 877~ 832w 740w

1031 w 1004w 937 mw, br 870 w, br 750 w 491 VW,vbr

v(CC) skeletal ci.~conformation v(CC) aromatic ring p(CHa) terminal PWW

6(CCH) aliphatic p(CH*) in phase VW)

assessment and evaluation of the effect of drug permeation and chemical treatment of skin for drug delivery enhancement. In a parallel study [20] we have reported our preliminary results for a similar study of human skin samples, which are subject to difficulties in sample standardization and to biohazard controls. Comparisons are currently being made between the vibrational spectra of human and reptilian skin samples and these will form the subject of a future paper [21].

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Acknowledgements-We are grateful to Drs P. Turner and A. Whitiey of Bruker Spectrospin Ltd, Coventry, for their assistance in obtaining the FT Raman spectra of the snake skins studied in this work.

REFERENCES [l] [2] [3] [4] [S] [6] [7] [8] [9] [lo] [ll] [12]

P. C. Rigg and B. W. Barry, J. Invest. Dermatol. 94,235 (1990). T. Itoh, R. Magavi, R. L. Casady, T. Nishihata and J. H. Rytting, Phurmuc. Res. 7, 1302 (1990). J. B. Roberts, J. Toxicol., Cut. Ocular Toxicol. 5, 319 (1986). T. K. Barajee and A. K. Mittai, j. Zool. (Lond.) 185,415 (1978). S. I. Roth and W. A. Jones, J. Ultrastruct. Res. 32,69 (1970). N. T. Yu, T. S. Lin and A. T. Tu, 1. Biol. Chem. 250, 1782 (1975). N. T. Yu, B. H. Jo and D. C. O’Shea, Archs Biochem. Biophys. 15671 (1973). A. T. Tu, B. H. Jo and N. T. Yu, Int. 1. Pept. Prot. Res. 8,337 (1976). L. Landman, 1. Morphol. 162, 93 (1979). L. Landman, 1. Ultr~truct. Res. 72,245 (1980). A. C. Williams, H. G. M. Edwards and B. W. Barry, ht. /. Pharmuc. 81, Rll (1992). J. G. Gras&h, M. K. Snavely and B. J. Bulkin, Chemicul Applications of Raman Spectroscopy. J. Wiley, New York (1981). [13] N. B. Colthup, Interpretation of Infrared Spectra. ACS Course, American Chemical Society, Washington (1971). [14] F. R. Dollish, W. G. Fateley and F. F. Bentley, Characteristic Ramun Frequencies of Organic Compounds. Wiley-Interscience, New York (1974). [U] B. Schrader, Angew. Chem. 12,884 (1973). [16] D. Yau, Ph.D. thesis, University of Bradford (1984). [ 171 P. R. Carey, Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York (1982). [18] H. H. Manstch, W. K. Surewicz, A. Muga, D. J. Moffatt and H. L. Casai, 7th International Conference on F. T. Spectroscopy (Edited by D. G. Cameron), Proc. SPIE 1145,580 (1989). [19] B. W. Barry, H. G. M. Edwards and A. C. Williams, 1. Ramun Spectrosc., 23, 641 (1992). [20] A. C. Williams, B. W. Barry and H. G. M. Edwards, Pharmac. Res. 9, S186 (1992). [21] A. C. Williams and H. G. M. Edwards, Proc. XXVIII Coil. Spectrosc. Internat., York (1993) to be published.