364 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Glazer and Smith (4) employed this reaction to estimate the amount of half-cystine in proteins. However, the reaction rate constants (kl, k2) and the equilibrium constant (K = k•/k2) were not determined. Without these parameters, multiple experiments with several concentrations of a protein were required to determine the cystine content. Herewith we are reporting a study on the exchange reaction of cystine in hair keratin hydrolysates and bis-DNP(dinitrophenyl) cystine to obtain the equilibrium and reaction rate constants. These constants in conjunction with the equilibrium or the kinetic data are used to determine the cystine contents in virgin and oxidized (bleached) hair fibers. METHOD MATERIALS Analytical grade chemicals were used in the study without further purification. Bis- DNP-cystine was obtained commercially. Brown Caucasian hair was used as the source of hair keratin. The hair was cleaned by twice shampooing and repeated rinsing with distilled water. All glassware was acid washed and rinsed well with distilled water prior to use. EXPERIMENTS A known amount of hair, ranging from 0.05 to 0.5 g, was digested in 25 ml of 12 N HCI at 50øC for 48 hours. Then the hydrolysate was filtered using a Milipore glass filter and diluted to 250 ml with 9.6 N HC1. A 20-ml aliquot was subsequently transferred to a screw cap vial containing bis-DNP-cystine. The concentration of bis- DNP-cystine used ranged from 1.2 to 10 mg per ml of hair hydrolysate. The vial was wrapped in aluminum foil to protect it from exposure to light and placed in a 39 + 0.5øC water bath. At certain time intervals, i.e., 1, 2, 3, 4, 8, 10, 15, 23, and 30 days, 1 ml of the reaction solution was withdrawn and diluted with 1.5 ml water. The unreacted bis-DNP-cystine in the solution was removed by extraction with 3 ml ethyl ether 6 times. The aqueous phase was quantitatively transferred to a 25-ml volumetric flask and diluted with 6N HCI. The concentration of mono-DNP-cystine in the solution was determined spectrophotometrically at 357 nm using a Cary 17D spectrophotom- eter. A sample of the hydrolysate without the reagent was taken through the complete procedure as the control. All experiments were run in duplicate. OXIDATION OF HAIR Hydrogen peroxide was used to bleach hair. Two solutions were prepared for the experiment. Solution A contained 10 g urea, 3 g glycerine, 7 g sodium chloride and 12 g ammonia hydroxide (30%) in 68 ml of distilled water. Solution B was a 6% (by weight) hydrogen peroxide solution. A mixture of 10 ml of each solution was heated to 32 -+ 0.5øC in a glass beaker. Then a 2 g hair tress was immersed in the mixture for 30 minutes. The temperature of the solution was maintained by a water bath and the solution was agitated throughout this period. The oxidized hair tress was rinsed thoroughly with distilled water and dried. DETERMINATION OF LIQUID RETENTION To determine the percentage of liquid retained by hair, a 0.2 g hair tress was immersed in a pH 7.00 buffer with 2 drops of 1% Triton X 100 solution. The hair was left in

HAIR BIS-DINITROPHENYL CYSTINE INTERCHANGE 365 the solution for 30 minutes. Then it was transferred to a centrifuge tube and centrifuged for 10 minutes at 1000 G. The tube had a wire mesh platform in the middle section to prevent the centrifuged hair from mixing with the liquid. Immediately following the centrifugation, the weight of the hair sample was determined. Then the hair sample was dried in a 105øC oven for 2 hours. When the sample was cooled, it was weighed again. The percentage of liquid retained was calculated from the difference of wet and dry weights divided by the dry weight. RESULTS AND DISCUSSION DETERMINATION OF THE EQUILIBRIUM CONSTANT The disulfide interchange reaction is very slow in hydrochloric acid having a concen- tration below 9N (2). In determining the concentration of Mono-DNP-cystine in the reacting solution, the acid concentration was diluted to about 2.4N during extraction. Therefore, because the reaction was stopped in the separation process, the concentration of mono-DNP-cystine should not be affected by removal of bis-DNP-cystine during the procedure. The reaction reached equilibrium in about 20 days. At equilibrium, the concentration of each species in the reacting solution may be expressed by [C] 2 k• - - K (eq. 2) [A][B] k2 where kx and k 2 are the forward and reverse reaction rate constants and K is the equilibrium constant. The other parameters, A, B, and C, are species shown in eq. 1. Under the chosen conditions, the solution was saturated with bis-DNP-cystine (B) throughout the experiment the concentration of B was constant at 1.3 m mole/1 (4). From the law of mass conservation, the concentration of cystine ([A]) is [A] = [A]0 - 0.5[C] (eq. 3) where [A]0 is the concentration of A at t = 0, i.e., the amount of cystine in hair keratin. It may be rewritten as [A]0 = m[Cy] (eq. 4) where [Cy] is the amount of cystine in ! g of hair and m is the weight of hair per liter of hydrolysate. Eq. 2 can then be reduced to [C] 2 = K'(m[Cy] - 0.5[C]) (eq. 5) where K' = K[B] (eq. 6) The solution for eq. 5, i.e., the concentration of mono-DNP-cystine at equilibrium, would be [C] = 0.5(-0.5K' + Q) (eq. 7) where Q = (0.25K '2 4- 4K' m[Cy]) •/2 (eq. 8)