OXIDATIVE TREATMENTS OF HAIR KERATIN FILMS 19 Figure 1. Solubility from untreated, hydrogen peroxide-treated, or bleach-treated hair keratin fi lms by var- ious solutions and SDS-PAGE analysis. Upper panel (A): Hair keratin fi lms were incubated with distilled water (UF), 1% hydrogen peroxide solution (HF), and commercial bleach mixture (BF). After washing and drying, the fi lms were incubated with various solutions at 50°C for 3 hr for protein solubility. A: 50 mM Tris- HCl (pH 8.5) (solution A). B: 50 mM Tris-HCl (pH 8.5) containing 8 M urea (solution B). C: 50 mM Tris- HCl (pH 8.5) containing 5 mM DTT (solution C). D: 50 mM Tris-HCl (pH 8.5) containing 8 M urea and 5 mM DTT (solution D). Lower panel (B): The protein components were analyzed by 5–20% SDS-PAGE. fi lms were incubated with solution D, containing urea and DTT, most of the proteins dissolved and the insolubility of the hydrogen peroxide-treated fi lms decreased. CHARACTERIZATION OF HYDROGEN PEROXIDE-TREATED KERATIN FILMS Urea is known as a typical denaturant that can destroy protein structures. In the absence of DTT, the protein dissolution from the untreated fi lm was dependent on the concentration

JOURNAL OF COSMETIC SCIENCE 20 of urea, while the protein dissolution from the hydrogen peroxide-treated fi lms increased only slightly as urea increased from 0 to 8 M (Figure 2A). Next, we examined the effects of hydrogen peroxide concentration on the protein dissolu- tion from the keratin fi lms (Figure 2B). The amount of proteins dissolved in solution B began to decrease when the concentration of hydrogen peroxide was more than 0.001%. Dissolved proteins from the fi lms decreased linearly when the fi lms were treated with increasing concentrations of hydrogen peroxide. The half-value was estimated to be ca. the 0.01% hydrogen peroxide solution, indicating that the hair protein fi lm was highly sensitive to the oxidative processing. MORPHOLOGY OF THE KERATIN FILMS WITH VARIOUS TREATMENTS The keratin fi lms were incubated with 1% hydrogen peroxide or the bleaching agent at 25°C for 10 min. The fi lms remained opaque irrespective of the hydrogen peroxide and the bleaching treatments (Figure 3A, panels a, b, c). However, the color was changed to the naked eye, from light brown to white, by the bleaching treatment, while the hydrogen Figure 2. Effects of urea and hydrogen peroxide on protein solubility from the hair keratin fi lms. (A) The keratin fi lms treated with distilled water ( ) and 1% hydrogen peroxide ( ) were incubated with solution A containing 0–8 M urea. The protein concentrations of the supernatants were measured. (B) The keratin fi lms were treated with 0.001–10% hydrogen peroxide and the solubility of solution B was examined.