J. Cosmet. Sci., 60, 527–535 (September/October 2009) 527 Enzymatic approach to analyze the effects of mercaptans on hair CHIKARA YAMAUCHI, AKIMASA MOCHIZUKI, KEISUKE TAKAYAMA, SATOSHI SUZUKI, and ATSUSHI SAKAINO, Higashi-Matsuyama Laboratories, Arimino Co. Ltd., 25-5 Miyako, Namekawa-machi, Hiki-gun, Saitama 355-0812, Japan, and WATARU OKAZAKI, Department of Life Sciences, Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan. Accepted for publication February 11, 2009. Synopsis Different mercaptans were used to prepare reduced hair and permed hair samples. The reduced hair showed much higher protease degradation than the permed hair also, the protease degradability of permed hair in- creased with repetitive treatments. The degradability of reduced hair was closely related to its cystine con- tent, and that of permed hair was nearly unaffected by its cystine content. The degradability of both the reduced and permed hair was related to the reduction power of each mercaptan used on the hair and increased in the following order: CYS-treated hair CA-treated hair TG-treated hair. INTRODUCTION In a permanent wave treatment, the hair-waving technique is a two-step operation involving the breaking of disulfi de bonds in hair using a mercaptan, and then regenerat- ing these bonds using an oxidizing agent (1). To evaluate the effect of mercaptans, many studies have been conducted (2–5). Morphologically, hair consists of a cuticle layer that envelops the fi brous hair cortex, constituting the bulk of the fi ber (6). The cortex mainly consists of two types of proteins: about 60% of intermediate fi lament proteins and about 40% of interfi brillar-associated proteins, traditionally called microfi bril (Mf) and matrix (Ma), respectively. The Mf is crystalline protein that is mainly composed of an α-helical protein with low cystine, and the Ma is an amorphous globular protein with high cystine (7,8). Recently, some investigators examined the effect of permanent waving on hair using a biochemical method, and reported interesting fi ndings about structural change in hair protein (7,9). We have also reported the correlation between the extent of protease degra- dation and the frequency of TG permanent wave treatments (10). Additionally, we showed that the degradation is closely related to the denaturation of Mf protein (10,11).

JOURNAL OF COSMETIC SCIENCE 528 In this study we examine the protease degradability of hair samples reduced with differ- ent mercaptans. The following mercaptans were used: thioglycolic acid (TG), cysteamine (CA), and cystein (CYS). After reduction the hair samples were either blocked with iodo- acetate or oxidized to reform broken disulfi de bonds. EXPERIMENTAL MATERIALS Fifty percent ammonium thioglycolate, 50% cysteamine hydrochloride, and l-cystein were employed as mercaptans, which consisted of thioglycolic acid (TG), cysteamine (CA), and cystein (CYS), respectively. The mercaptans, 28% ammonium hydroxide, and so- dium bromate were of cosmetic grade, and a special reagent grade sodium dodecyl sulfate (SDS) was used without further purifi cation. Pronase E was supplied by Sigma (St. Louis, MO) for Streptomyces griseus, 12.4 U/mg. All other chemicals used were of reagent grade. The untreated hair (71 μm average diameter) used was from Chinese women in their 20s who had never had chemical hair treatments. The hair samples were soaked in 1 wt% SDS aqueous solution for 10 min at 25°C, washed with water for 30 min, and air dried. PREPARATION OF REDUCED HAIR AND PERMED HAIR Reduced hair. Reduced hair samples were prepared by reduction and subsequent blocking steps (12). About 1.0 g of untreated hair was soaked for various lengths of time in 0.50 M mercaptan aqueous solution, as shown in Table I, using a 10:1 solution-to-hair ratio at 25°C. The hair was then removed and immediately soaked for 5 min in 1 wt% ice-cold iodoacetic acid aqueous solution, using a 100:1 solution-to-hair ratio. The hair was then treated for 45 min in 1 wt% iodoacetic acid aqueous solution (adjusted to pH 8.4 with sodium hydroxide), using a 100:1 solution-to-hair ratio at 80°C, to effect blocking of the SH groups. The hair was then washed with water for 30 min and air dried. Permed hair. Permed hair samples were prepared by reduction and subsequent oxidation steps (10). About 1.0 g of untreated hair was soaked for various lengths of time in mer- captan aqueous solutions, as shown in Table I, using a 10:1 solution-to-hair ratio at 25°C. The hair was immediately soaked for 15 min in an oxidizing agent, as shown in Table I, Table I Permanent Waving Solutions Mercaptan solutions Oxidizing agent TG CA CYS Ammonium thioglycolate 0.50 M — — — Cysteamine hydrochloride — 0.50 M — — L-Cystein — — 0.50 M — Ammonium hydroxide pH 8.6 pH 8.6 pH 8.6 — Sodium bromate — — — 0.4 M Phosphoric acid — — — pH 4.0 Deionized water to 1000 ml to 1000 ml to 1000 ml to 1000 ml