CHEMISTRY OF HUMAN HAIR CUTICLE 365 cystine in keratins by thioIs is difficult to achieve because the reaction is determined by the law of mass action, dithiothreitol reduction readily splits 85• of the disulphide bonds in wool under mild conditions with only a small excess of dithiothreitol (14, 15) owing to the formation of a stable cyclic disulphide (4,5-dihydroxy-l,2-dithiane). After reduction of the disul- phide bonds the proteins, previously resistant to attack by proteolytic enzymes, are rapidly digested under mild conditions. Borenfreund, Fitt and Berdich (17) have used combinations of trypsin and reducing agents including 2-mercaptoethanol and 2,3-dimercaptopropanol to degrade 'proteolytic enzyme resistant, keratin-like' components of sperm cells and similarly Pfau and McCrea (18) have used pronase and 2-mercaptoethanol to release DNA from vaccinia virus. Preliminary experiments by us have shown that a combination of pronase and dithiothreitol rapidly digests human hair cuticle (80•o being dissolved in 1-} h at 37øC pH 8.0 and over 91• within 16 h) indicating that pronase may prove to be a valuable alternative to the use of papain at 50øC. The activation of papain is dependent upon the existence of cysteinyl residues at the prosthetic site (16) so that the presence of dithiothreitol will give maximum yield of the groups and thereby maximum proteolytic activity. ACKNOWLEDGMENTS We are grateful to Mr F. J. Bailey of Unilever Research Laboratory, Colworth House for undertaking amino acid analyses for us. Thanks are also due to Mrs S. J. Smith for her valuable assistance with the electron microscope work. (Received: 28th February 1974) REFERENCES (1) Swift, J. A. and Bews, B. The chemistry of human hair cuticle. Part 1. A new method for the physical isolation of cuticle. J. $oc. Cosmet. Chem. 25 13 (1974). (2) Fraser, R. D. B., Macrae, T. P. and Rogers, G. E. Keratins: Their Composition, Structure and Biosynthesis (1972) (Charles C. Thomas, Springfield, Illinois). (3) Bradbury, J. H. and Ley, K. F. Separation and analysis of exocuticle and endocuticle. Aust. J. Biol. $ci. 25 1235 (1972). (4) Swift, J. A. and Bews, B. The isolation of membranes from keratin fibres with papain and dithiothreitol. J. Text. Inst. 65 222 (1974). (5) Spurr, A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct, Res. 26 31 (1969).

366 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (6) Reynolds, E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17 208 (1963). (7) Swift, J. A. The electron histochemistry of cystine-containing proteins in thin transverse sections of human hair. J. Roy. Microsc. Soc. 88 449 (1967). (8) Bradbury, J. H. and Leeder, J. D. The cell membrane complex of wool. Appl. Polymer Syrup. 18 227 (1971). (9) King, N. L. R. and Bradbury, J. H. The chemical composition of wool V. The epicuticle. Aust. J. Biol. Sci. 21 375 (1968). (10) Kiihn, K., Grossman, W. and Hofman, U. Die elektronenmikroskopische anffirbung des kollagens und die ausbildung einer hochunterteilen querstreifung. Z. Naturforsch. 136 154 (1958). (11) Dobb, M. G., Murray, R. and Sikorski, J. Specific labelling of thiol groups in mammalian keratin suitable for electron microscope studies. J. Microsc. 96 285 (1972). (12) Levy, D. The selective chloromercuration of insulin. Biochim. Biophys. Acta 317 473 (1973). (13) Swift, J. A. The electron cytochemical demonstration of cystine disulphide bonds using silver-methenemine reagent. Histochemie 35 307 (1973). (14) Weigmann, H-D. and Rebenfeld, L. Reduction of wool with dithiothreitol. Text. Res. J. 36 202 (1966). (15) Weigman, H-D. Reduction of disulphide bonds in keratin with 1,4-dithiothreitol. Part 1. Kinetic investigation. J. Polym. $ci. 6 (AI) 2237 (1968). (16) Glazer, A. N. and Smith, E. L. Papain and other plant sulphydryl proteolytic enzymes. In: The Enzymes Part 3, ed. P. D. Boyer, 501 (1971) (Academic Press, New York). (17) Borenfreund, E., Fitt, E. and Berdich, A. Isolation and properties of deoxyribonucleic acid from mammalian sperm. Nature, 191 375 (1961). (18) Pfau, C. J. and McCrea, J. F. Release of deoxyribonucleic acid from vaccinia virus by 2-mercaptoethanol and pronase. Nature, 194 894 (1962).