CYSTEINYL AND S-SULFOCYSTEINYL RESIDUES Time of Storage (days) Table II MNP• Titer of Sulfite-Treated Hair Samples After Storage in 6N H2SO• • Ker-CySH q- Ker-CySSOa- meq/g hair (MNP•) 467 0 (control) 0.81 2 0.81 5 0.82 7 0.82 a 1.0M NH4HSOa-3M urea, pH 5.8, 32øC, 50:1 bath ratio for 30 min. Table III Effect of 6N H_oSO• Rinsing on Ker-CySH Level of Thioglycolate-Reduced Hair as Determined by the Iodoacetamide Procedure s meq SH/g Reduction Time b Before After (minutes) Rinsing Rinsing 5 0.25 0.23 10 0.41 0.41 Two 1-min and one 3-min rinses with isopropanol followed by two 1-min and one 13-min rinses with 6N H•SO4. 0.30N ammonium thioglycolate, pH 9.2, 32øC, 50:1 bath ratio. achieved in the keratin-bisulfite mixture? We have assumed not in the absence of any clear experimental data to the contrary. That the acid rinse does not change the reduction level of keratin treated with mercaptan is verified in the experiment in which thioglycolate-treated hair was analyzed by the iodoacetamide procedure (2) before and after acid rinsing, as shown in Table III. The remainder of assumption (a), that acid rinsing desorbs soluble mer- captan from keratin, was demonstrated on thioglycolate-treated hair. Using only the acid rinse sequence described in Table III, no sulfhydryl content, as determined by nitroprusside, could be found in the second and third acid rinses. Pertinent to assumption (b), the alkaline reversibility of the cystine-bi- sulfite reaction, is the fact that rinsing bisu]fite-treated keratin with an alkaline buffer causes a lowering of the MNP• titer of the fiber as shown in Fig. 2. Our method is based on the assumption that exactly one-half of the titratable groups lost during alkaline reversal are CySSO.•- groups and the other half are CySH groups. This assumption is supported by Wolfram's

468 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 75 / , I / I i o 5 lO Time (minutes) Figure 2. MNP• titer of bisulfite-treated hair as a function of pH 10 rinse time. Treatment conditions: 1.0 M NH4HSO•-3M urea, pH 6.6, 32øC, 3:1 bath ratio for 30 min finding that the decrease of MNP• titer in an alkaline reversal process is balanced by an increase in 'the fiber cystinc content (6). The data presented in Table IV show that CySH groups alone are not lost during alkaline reversal that is, MNP• is equal to MNP,. for hair con- taining only eysteinyl residues. To examine this point further, we prepared hair containing only CySSO3- residues using the oxidative sulfitolysis procedure with bisulfite-tetrathio- hate (7). The data presented in Table V also show that CySSO3- groups alone are not lost during reversal for hair containing only these residues that is, MNPt = MNP2 = MNP,•. Since, under the conditions of alkaline re- versal, the disappearance of either CySSOa- or CySH residues does not occur unless both spedes are present (and when it does occur it is balanced by an increase in the fiber cystinc content), it is difficult to envision a mecha- nism of reversal other than that described for the classical eystine-bisulfite reaction. That is, one fiber CySSO:•- residue combining with one fiber CySH residue to yield one fiber cystinyl group. The third assumption on which our method is based is that acrylonitrile alkylation of alkali reversed keratin quantitatively converts cysteinyl me- captan to fi-cyanoethylsulfide without loss of CySSOa- residues. The validity