Chapter 1 11 abutting the epicuticle (c.f. Figure 4). Its intense staining with ammoniacal silver nitrate signifies its proteins are particularly rich in cystine. Using an unusual method of electron-probe X-ray microanalysis it has been established that the first 1000 Å at the hair surfaces, of which the A-layer constitutes the major part, contains approximately 11.8 weight % of sulphur.7 From this work it was calculated that proteins of the A-layer thereby contain approximately 1 in 2.7 of their amino acid residues as ½ cystine residues. This makes for a truly remarkable protein. Its extreme toughness would seem to be assured if all the cystine is engaged in intermolecular disulphide cross-links and fitting for this component to be located close to the hair surface to withstand mechanical insults. The A-layer is remarkably resistant to attack by proteolytic enzymes.28, 29 Using papain in conjunction with dithiothreitol, this reagent was shown to be capable of removing most of the proteins from the hair fiber in super-quick time28, whereas the cuticular A-layer remained relatively intact and only dissolved slowly with much longer term treatment. Resistance to digestion might have been due to the extraordinarily high cystine of the A-layer but might also have been due to the underlying presence of intermolecular isodipeptide bonding in the proteins. The amino acid composition of the A-layer was quoted in one of these works28 but this is now thought to be in considerable error. Thus the ½ cystine content of the enzyme- derived material was determined to be 99 amino acid residues per 1,000, whereas later analysis by a purely physical analytical approach7 showed a ½ cystine content to be as high as 370 residues per 1,000. One suspects the presence of dithiothreitol in the digestion system had led to specific loss of cysteine-rich material. Interestingly this might have been due to the presence of small peptides, such as the dipeptide cysteinyl cysteine having been present as a cross-link with protein main chain cysteine residues. In partial support of this idea, it is worth noting Buchanan30 identified the presence of the dipeptide cysteicyl cysteic acid in solution following the oxidation of human hair with performic acid. No further work has been reported in this area.