J. Soc. Cosmet. Chem. 25 355-366 (1974) ¸ 1974 Society of Cosmetic Chetnists of Great Britaht The chemistry of human hair cuticle- II: The isolation and amino acid analysis A-layer of the cell membranes and J. A. SWIFT and B. BEWS* Synopsis--Mixtures of papain and dithiothreitol have been used to effect the separation of the A-LAYER and CELL MEMBRANE COMPLEX and the cell membrane complex alone from human HAIR CUTICLE. The course of these ENZYMATIC DIGESTIONS has been followed gravimetrically and by the ELECTRON MICROSCOPE examination of digested hair sections, and it is evident that the components mentioned are isolated cleanly. The AMINO ACID com- positions of the cell membrane complex and of the A-layer (obtained by difference) are quite different from the composition of the whole cuticle. The significance of the analyses is discussed. INTRODUCTION The previous paper of this series (1) described a method for the physical isolation of cuticle from human hair and emphasized the lamellar substructure of each cuticle cell sheet. Our interest is now in the further separation of these laminae for chemical analysis. Various chemical pro- cedures have been described for isolating different fractions from keratin fibres (cf. reference 2). Many of these lead to uncertainties about the signi- ficance of the subsequent chemical analysis and most do not allow the * Unilever Research Isleworth Laboratory, 455 London Road, Isleworth, Middlesex. 355

356 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS accurate specification of the morphological origin of the isolated fractions. Enzymic digestion procedures, by virtue of their high chemical specificity, are likely to lead to much cleaner fractionation and indeed Bradbury and Ley (3) have recently used pronase to dissolve wool endocuticular com- ponents selectively. Using mixtures of papain and dithiothreitol we have shown (4), by the electron microscope examination of digested hair sections, that a distinct layer remains undissolved in the vicinity of the cuticle cell membranes. The present paper is concerned with the further study of this digestion procedure which has enabled us to separate from isolated human hair cuticle the cell membrane complex and A-layer. METHODS AND MATERIALS The isolation of human hair cuticle This material was prepared by shaking root-end pieces of brown Caucasian hair in water according to procedures described in the previous paper of this series (1). Cuticle fragments obtained after 2 h shaking (i.e. 5• by weight of the original hair) were separated and dried in vacuo over phosphorus pentoxide. Digestion of cuticle fragments with papain/dithiothreitol Preliminary gravimetric experiments, in which isolated cuticle was digested by dispersion in a solution containing I mg/ml papain (crude powder type 2, Sigma) and 2 mg/ml dithiothreitol (Calbiochem) in 0.1M phosphate buffer at pH 6.7, showed that 92•o of the cuticle was dissolved after 18 h at 65øC. In addition, from observations in the transmission elec- tron microscope of thin transverse hair sections digested under similar conditions, it was evident that some components were almost completely dissolved in as little as 15 min at 65øC. The rate of digestion could be reduced by working at 50øC and this lower temperature was used in all subsequent experiments. The course of the digestion was studied by using the following procedure. Weighed samples of cuticle (30 mg) were triturated with phosphate buffer (0.1M, pH 6.7) to form a uniform suspension. A solution of papain (2.5 mg) and dithiothreitol (10 mg) in buffer (2 ml) was then added and the suspensions incubated in a water bath at 50øC. After appropriate time intervals, samples were withdrawn and chilled to quench the enzyme reaction. The undigested cuticle residues were recovered by