MELANIN OF HUMAN HAIR 143 Table V Hair Sensitization by Treatment with LiBr Solutions Time % Hair Digested Source of Pigment (min) by the Enzyme Italian Brown Hair 60 85 Japanese Black Hair 90 90 Irish Red Hair 60 91 Scandinavian Blond Hair 60 93 2. Hair digestion. PSF 2019 is a powder which consists of 35% protein. Mineral salts are present, chiefly ammonium sulfate. Although such raw material is active on sensitized hair, we prefer to purify the enzyme, in order to avoid contamination of the melanin granules. A solution of 2 g of raw proteinase in 100 ml of water is dialysed for 24 h, against demineralized water at 4øC. The solution becomes more acid (final pH 5.3) and a fraction of the enzyme is insolubilized. When the enzymatic solution is not used immediately, the solution is lyophilised. The resulting lyophilised powder now contains 80% protein and only very small quantities of ammonium sulfate. The aminoacid composition of purified proteinase is given in Table VI. Table VI Amino Acid Composition of Purified Proteinase PSF 2019 (mMoles per 100 g of protein) Aspattic Acid 11.0 Threonine 8.1 Serine 8.7 Glutamic Acid 7.8 Proline 2.8 Glycine 11.6 Alanine 9.2 Cystine 0 Valine 6.0 Methionine 0 Isoleucine 2.9 Leucine 4.7 Tyrosine 1.6 Phenylalanine 2.4 Lysine 2.1 Histidine 1.5 Arginine 3.3 The total proteolytic activity of lyophilised PSF 2019 is determined by the Anson method (31) on hemoglobin substrate. Its activity is 16,500 Anson units/mg of powder. a) Conditions of digestion. Buffer.' The amount of hair digested depends on the pH of the enzyme solution (Figure 1). The alkaline pH (9.0) is obtained by a Borate-buffer

144 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ,oo Z 90X 70X \ Amount of digested hair , , , , , .,,•pH 6.0 7. 0 8.0 9.0 10.1• Figure 1. Influence of pH on the digestion of Italian brown hair by the enzyme. Sensitization: DMF treatment. Digestion temperature: 40øC. (S 5rensen). This buffer is boiled before use, in order to avoid the solubilization of NH 3. Temperature.' Temperature is maintained at 40øC. At 50øC, digestion is quicker but the total amount of digested hair slightly decreases, because of the autolysis of the enzyme, and the risk of chemical transformations of hair protein during digestion increases. We checked that our conditions of digestion, pH 9.0 at 40øC caused no transformation of cystine in the dissolved protein to lanthionine. Stirring.' Continuous stirring is necessary in order to increase the amount and the kinetics of digestion. A double-bladed propeller connected to an electric motor ensures a slow but continuous stirring of the enzyme solution. Amount of enzyme: We studied the yield of digestion in terms of enzyme/substrate ratio (E/S) (Figure 2). Enzyme is expressed in weight of total protein content of purified lyophilised PSF 2019. At E/S • 1.5, 100 g of sensitized hair are digested by 1.44 g of purified lyophilized proteinase PSF 2019 (expressed in weight of total protein content). Time of digestion.' We followed digestion during 5 days (Figure 3). After 4 days digestion, proteinase has lost its activity on hair substrate. If a second equivalent dose