EFFECTS OF MERCAPTANS ON HAIR 529 using a 10:1 solution-to-hair ratio at 25°C. The hair was not rinsed before neutralization, since this was found to cause reoxidation. Then the hair was washed with water for 30 min and air dried. This process was repeated to prepare damaged hair samples, with a reduction time fi xed at 15 min. AMINO ACID ANALYSIS Hair samples were hydrolyzed in 6 N HCl at 105°C for 24 hr in a nitrogen atmosphere. After membrane fi ltration of the solution, the acid and water were removed on a rotary evaporator at 45°C, and phenylthiocarbamyl (PTC) derivatization of the hydrolysates was performed using the method of Bennett and Solomon (13). The PTC derivatives were analyzed on a Jasco HPLC system consisting of a PU-980 pump, a DG-980-50 degasser, an LG-980-02 gradient unit, a CO-965 column oven, and a MD-910 multi-wavelength detector set at 230 nm. The system was delivered in an isocratic and subsequent gradient mode to an Inertsil ODS-2 column (100 mm × 4.6 mm i.d., GL Science Inc.), as described in a previous article (14). The degree of cystine (disul- fi de bonds) reduction of hair was calculated as follows: R R R R 1 (%) [( )/ ] = - ´ 0 1 100 where R is the percentage of cystine reduction, and R1 and R0 are the amounts of cystine in untreated hair and reduced or permed hair, respectively. The value represents an aver- age of the result of triplicate experiments. PROTEASE TREATMENT Hair samples were cut to 2.0-cm length, and about 100 mg of the hair sample was incu- bated in 2.0 ml of Tris-HCl buffer (20 mM, pH 8.0) containing 0.05 wt% Pronase E, or without addition of Pronase E (control), at 37°C for 100 hr. After centrifugation of this solution at 9,000 rpm for 5 min, the supernatant was discarded. The residual hair was washed with water, and after centrifugation of this solution, at 9,000 rpm for 5 min, the supernatant was discarded. This process was repeated three times. After drying, the resi- due was weighed and the degree of degradation was calculated as follows: P P P P (%) [( )/ ] = - ´ 1 0 1 100 where P is the percentage of the extent of degradation, and P1 and P0 are the weights of hair before and after Pronase E treatment, respectively. The value represents an average of the result of triplicate experiments. WATER RETENTION Water retention was measured using a method similar to that of Kohara et al. (15). Hair samples were immersed in deionized water for 4 hr at 25°C, then centriguged at 3000 rpm

JOURNAL OF COSMETIC SCIENCE 530 for 5 min and weighed (W1). After drying for 3 hr at 105°C, the hairs were weighed (W0) and the degree of water retention calculated as follows: W W W W 1 (%) [( )/ ] = - ´ 0 0 100 SCANNING ELECTRON MICROSCOPY (SEM) Hair samples were cut at their edges with scissors, and part of these samples was treated with the protease described above. The hair samples before and after Pronase E treatments were mounted on stainless steel stubs and sputtered with gold (10 mA, 5 min). The mor- phology of the hair was examined using a Jeol JSM-5200 scanning electron microscope (accelerating voltage: 15 kV magnifi cation × 1000). RESULTS AND DISCUSSION EFFECT OF REDUCTION TIME ON HAIR First, reduced hair and permed hair samples were prepared using different reduction times then the extent of degradation of the hair with protease was measured. As shown in Figure 1, the degradation extent of each mercaptan-reduced type of hair increased with increasing reduction time. It appears that the effi cacy of protease increased due to the cleavage of disulfi de bonds (cystine) in hair. Furthermore, in the entire treatment time, the degradation extent of CYS-reduced hair was the smallest of all the reduced hairs, that of CA-reduced hair was much higher, and that of TG-reduced hair was the greatest. The differences in the extent of degradation may due to the different extents of cystine cleav- age in the hair. For permed hair, the degradation extent of each hair also increased with increasing reduc- tion time (Figure 2). The difference in the extent of degradation between the permed hair was smaller than that of reduced hair. It appears that the oxidation treatment of permed hair affects the protease degradation. In the oxidation treatment of the permanent waving Figure 1. Correlation between the degradation extent and the reduction time of reduced hair. The reduction time of the reduced hair is 5, 10, 15, 20, and 30 min.