26 JOURNAL OF COSMETIC SCIENCE That the A-layer was relatively resistant to digestion by mixtures of papain and dithio- threitol (15), under conditions in which almost all the other hair's components dissolve, was the first indication that perhaps this structure contained isodipeptide crosslinks in addition to the large amounts of cystine disulphide crosslinks already known to be present. Such crosslinks occur as a protein intermolecular peptide bond, ½-amino-(•/- glutamyl) lysine, and their formation between lysyl and glutaminyl side chains in the follicle is facilitated by the calcium-dependent enzyme, transglutaminase (cf. Figure 3) (16). Fractions of cuticle derived by papain/dithiothreitol digestion, and containing the CMC and significant residues from the A-layer, were shown by Nienhaus (17) to possess isodipeptide at a level of ca. 70 pmol/g. Later Zahn eta/. (18) estimated a concentration of 2.5 mole% of isodipeptide for the enzyme-resistant material, based on the assumption that 50% of the 5 mol% of the protein's lysine residues had participated in isodipeptide formation. What remains uncertain is the distribution of these isodipeptide-rich pro- teins within the cystine-rich A-layer or indeed within the cuticle as a whole. An attractive notion is that they are concentrated in a superficial subcomponent of the i co Glutamine sine co I I HC"-- C-"- C-'- C'--- N H2+ H2N•-C•C•C---C CH I H H0 H H H H2 I NH NH ! ii ! ! ! ! I I I Transglutaminase +Ca 2+ (-NH3) C---- C • C-'----N---- C---- C--- C---- C---- CH isopeptide linkage H2 H2 H2 NH ! s-ami no-(l,-gl utamyl)lysine Figure 3. The formation of isopeptide intermolecular crosslinks. This occurs in some hair proteins by the action of transglutaminase upon spatially proximate glutamine and lysine sidechains.

HUMAN HAIR CUTICLE 27 A-layer. Thus, together with an external layer of covalently linked fatty acid (see below), this seems likely to comprise the thin membranous sacs (so-called epicuticle) specifically raised from the cuticular surfaces of all mammalian keratin fibers when they are treated with dilute chlorine water (19,20). Isodipeptide-rich proteins also comprise thin, in- soluble intracellular envelopes in other keratinized structures such as the stratum cor- neum (21). Rice et al. (22) have reported TEM observations of mammalian hairs, from which most proteins had been extracted with hot mixtures of sodium dodecyl sulphate and dithiothreitol, and found isopeptide-rich proteins as a thin intracellular envelope for all the hair's cells, including the cortex. Thus all the cells of the hair shaft seem to possess these thin insoluble envelopes, and it is highly likely on this basis that the whole intracellular surface of each cuticle cell, and not merely the A-layer, possesses such an envelope. The suggestion has been made (23) that, on the assumption the majority of the cystine residues of the A-layer proteins are engaged in intermolecular crosslinking, this will render the structure tough and fitting in its role of protecting the hair surface from mechanical insult. Such a highly crosslinked protein, one anticipates, would swell little in water (as compared with other structures in the hair shaft). It can be expected to possess a relatively high indentation hardness and a very high modulus of elasticity as compared with the components of the hair cortex. One also expects the A-layer to fracture under applied tensile stress at strain levels significantly less than for the hair as a whole, and indeed this could be the focus for catastrophic circumferential fractures through the cuticle sometimes seen in individually stretched hairs or in hair tresses multiply combed (24). There is the additional possibility that the A-layer and accom- panying exocuticle undergo fracture at relatively low levels of strain in the bending of the cell sheet. This could be an advantage in ensuring that only small pieces are shed from the cuticle scale edges by frictional interaction, thereby helping to extend the cuticle's lifetime in protecting the main bulk of the hair shaft. The A-layer provides support for the rigid attachment of covalently linked fatty acids at the hair's surface (see below), which undoubtedly aids the frictional performance at this surface. THE EXOCUTICLE This sheet-like component, which again is proteinaceous, smoothly abuts the A-layer, but its other side is highly sculptured. Its thickness, accordingly, varies between ca. 100 and 300 nm. Under the TEM the exocuticle appears to be amorphous. Electron histo- chemical experiments have shown it to be particularly rich in cystine, i.e., at a higher concentration than in the proteins of the hair cortex but not as high as in the A-layer (5). Differential protease digestion of physically isolated human hair cuticle has been used to derive an amino acid analysis for the proteins of the exocuticle (6). Approximately 20% of the amino acid residues were as •/5-cystines. That the structure is relatively devoid of isodipeptide crosslinks is indicated by the relatively high speed of its digestion with papain/dithiothreitol, a reagent that otherwise only slowly dissolves the adjacent isodi- peptide-rich A-layer. Individual proteins have not yet been specifically isolated from the exocuticle, and so we don't know how many different types there are, or their amino acid sequences or conformation. The proteins can be expected to belong to the classes of high- and ultrahigh-sulphur proteins commonly identified in polyacrylamide gel electropho- retic separations from solubilized whole fibers (10,12).