HAIR MELANIN 183 (14). In each case approximately 10 g of hair were used, and the hair was spread parallel and rearranged daily to assure uniform exposure to irradiation. COLOR DETERMINATION The color of the nonirradiated hair was determined by means of Datacolor © 3890 equipment using the CIELAB-system. The color of the irradiated hair was determined visually by comparing the color quality with nonirradiated light-brown and black tresses. ISOLATION OF MELANIN An enzymatic technique to solubilize the keratin was used (15). One gram of hair was stirred into 30 ml of buffer of pH 6.7 containing 83.36 mg papain and 290 mg dithioerythritol (DTE). The reaction was carried out for 72 h at 50 ø C. The residue was separated by centrifugation (18,000 min- • at 4 ø C), with twofold washing with water. The residue contains cell membrane complex and melanin granules. SEPARATION OF THE CELL MEMBRANE COMPLEX AND PROTEINS FROM THE MELANIN The isolated mixture containing the cell membrane complex and melanin granules was hydrolyzed for 120 h in vacuo at 110 ø C with 6 N HC1 and 3% thioglycolic acid. The melanin residue was filtered using a 0.2-}xm pore membrane. DELIPIDIZATION OF MELANIN The deproteinated melanin was delipidized by three 10-min washings with hexane/ isopropanol/water (6:6:1 v/v/v). The residue was freeze-dried and kept at P205. SEM EXAMINATIONS The purity of melanin was evaluated by means of a scanning electron microscope (SEM). IR SPECTROSCOPY The spectroscopic examination was carried out using an FTIR 60SXR spectrometer (Nicolet). The melanin samples were examined either as KBr pellets or in a diamond cell. The spectral resolution was 4 cm- 1 in the range from 400 to 4000 cm- 1 RESULTS IRRADIATION OF HAIR SAMPLES The black and light-brown hair samples were irradiated for a period of 6 weeks (1008 h) with UV-B, UV-A, visible light, IR, or global radiation as previously de- scribed (14).

184 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS COLOR MEASUREMENTS Before irradiation, color measurements were carried out using the CIELAB-system with notation of L* for lightness and a* and b* for the red and yellow values, respectively (Table I). As expected, the black hair shows a low L* value (L* = 17.3) and minimal a* and b*. The light brown hair is distinctly lighter (L* = 32.6), more red (a* = 6.9), and more yellow (b* = 14.4). Therefore, we suggest a contribution of the reddish-brown pheomelanin pigments to its original color. The photochemically induced color changes of black and light-brown hair were subjec- tively assessed and discussed in the first part of this publication (14). They are referred to in the present paper in Table II to complete the discussion about pigment alterations. The data in Table II indicate that the light-brown hair photobleaches more extensively than the black hair, a fact already observed in real life by others (16). However, it has become possible for the first time to assign the extent of these color changes to specific segments of the sunlight. The results clearly show that the photobleaching of hair is dependent on the initial color of the hair as well as on the segment of the sunlight spectrum. ISOLATION OF THE MELANIN PIGMENT After enzymatic dissolution of the keratin, the insoluble residue consists of the cell membrane complex (CMC) and melanin granules. Separation of the proteins was ac- complished by an acid hydrolysis. Thioglycolic acid was added to the hydrolysis mixture to prevent oxidation of the melanin. After 120 h of hydrolysis, followed by a deli- pidization and a micropore filtration, no fibrous contaminants derived from cells or the cell membranes were present in the granular samples. In that way SEM examinations attested to the satisfactory purity of the melanin obtained (Figure 1). The pigment granules isolated from black hair show a typical rice-like appearance, with a length of 0.7-1.0 p•m and a width of 0.4-0.5 p•m (Figure la). On the other hand, the melanin granules from the light-brown hair (Figure lb) are generally smaller and of irregular shape. Some are similar to those of black hair (0.65 p•m X 0.3 p•m). Most of them appear more circular and smaller (0.35 p•m to 0.46 p•m in length and 0.17 p•m to 0.23 p•m in width), which is characteristic of granules consisting of pheomelanin (1). This would suggest that the color of the light-brown hair is primarily due to the pheomelanin pigment and to a much smaller extent to eumelanin. EFFECT OF IRRADIATION OF HAIR ON THE CONTENT OF MELANIN It was found that 5-9 % of the melanin, including the CMC, could be isolated upon Table I Color Measurements of Nonirradiated Black and Light-Brown Human Hair Black 17.3 1.8 1.6 Light-brown 32.6 6.9 14.4