MELANIN OF HUMAN HAIR 145 lOO • 807, 7oZ 6o, \Amount of digested hair i ii i i i 0.36 0.72 1.08 1.44 1.80 10-2 10-2 10-2 10-2 10-2 E Figure 2. Influence of the enzyme/substrate (E/S) ratio on the digestion of Italian brown hair by the enzyme. Sensitization: DMF treatment. of new purified proteinase is added to the mixture, or if centrifugated undigested residue is again placed in a new solution of enzyme, no further digestion occurs with DMF sensitized hair, but with LiBr sensitized hair, digestion reaches about the same final amount (90%) as with DMF sensitized hair after the first digestion (Figure 4). b) Melanin characterization. Our experimentation showed that, after 3 HCl-hydrol- yses (according to our conditions), no protein may be found by further hydrolysis (Figure 5). Pigment samples at this stage of preparation have been denoted as pure melanin granules. All pigment samples have been examined by scanning electron microscopy (Figures 6 and 7). Pigments extracted from Italian brown hair and Japanese black hair show the typical rice grain shape, with granu'les extracted from Asian hair slightly larger. Pigments extracted from Irish red hair or Scandinavian blond hair, however, do not exhibit any organized structure. According to the usually accepted diagram of melanization, black polymer (eumelanin) is deposited on a protein matrix, and forms structured organelles which, at the first stages, are poor in melanin (premelanosomes). As the pigment deposition progresses, discrete compact melanin-containing melanosomes are formed. These appear uniformly brown when brown or black hair sections are observed under the electron microscope.

146 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 100Z 80Z 60/o 4OZ 20/, Amount of digested hair DMF treatment LiBr treatment {, Time I I I I I % I day 2days 3days 4days 5days Figure 3. Influence of time on the digestion of Italian brown hair by the enzyme. soY, 7oZ Amount of digested hair DMF treatment LiBr treatment I I I I end of the 1day 2days 3days first digestion Figure 4. Second digestion by the enzyme. Time , 4days