CYSTEINYL AND S-SULFOCYSTEINYL RESIDUES 469 Table IV MNP Analysis of Thioglycolate-Reduccd Hair '• Total cleavage (MNP•) After alkaline reversal (MNP,o) After alkylation (MNP.•) meq SH/g 0.• (- 0.,33 0.00 ().SON ammonium thioglyeolate, pH 9.9., $2øC, 50:1 bath ratio for 30 min. Table V Analysis of Hair Treated with Bisulfite-Tetrathionate" meq SH/g MNPx 0.87 MNP.o 0.87 MNPa 0.86 Ker-CySH 0.01 Ker-CySSO•- 0.86 30-min immersion in 1.0M NH•HSOo-4.0M urea-0.4M tetrathionate, pH 8.0, 50:1 bath ratio, 32øC. of this assumption is confirmed by two experiments. The data in Table IV show that for hair reduced with thioglycolate, the alkylation with aeryloni- h'ile is quantitative that is, MNPa = 0. The fact that alkylation does not destroy CySSOa- groups is demonstrated in Table V for the analysis of hair containing only CySSOa- groups. MNPo_ and MNPa are essentially the same, indicating that CySSO:•- groups are not consumed during the alkylation reaction. In the application of the method to hair containing predictable CySSO:•- to CySH ratios, the utility of this method depends on whether or not the method can quantitatively distinguish CySSOa- from CySH in keratin con- taining these residues in all proportions. There is ample evidence that this is so. A test of the validity of the method was obtained by performing the analyses on hair samples containing predictable ratios of CySSO.•- and CySH. Hair was modified as described in Table VI. Treatment with bisulfite afforded hair with equal amounts of cysteinyl and S-sulfocysteinyl residues. Reduction with thioglycolate followed by bisulfite yielded hair in which there were more CySH residues than CySSO.•- residues. Treatment xvith bisulfite and tetrathionate followed by treatment with bisulfite provided hair where the number of CySSOa- residues exceeded the CySH residues. As discussed previously, treatment with bisulfite-tetrathionate, fl•at is, oxida- rive sulfitolysis, yielded hair with CySSOa- residues only, while reduction with thioglycolate afforded hair with CySH residues only.

470 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table VI Preparation of Hair Containing Unequal Amounts of Ker-CySH and Ker-CySSOa- Condition Treatment Ker-CySH = Ker-CySSO•- Ker-CySH Ker-CySSO•- Ker-CySH Ker-CySSO•- Ker-CySSO•- only Ker-CySH only Bisulfite Thioglycolate followed by bisulfite Bisulfite-tetrathionate followed by bisulfite Bisulfite-tetrathionate Thioglyeolate Table VII Analysis of Hair Treated with Sulfite Systems a Immersion Ker-CySSOa- Time meq SH/g meq/g meq/g pH (hours) MNP• MNP• MNP3 Theor * Calc • Theor b Calc" 3.5 1.25 0.70 0.32 0.15 0.35 0.35 0.35 0.36 4.5 1.25 0.88 0.20 0.10 0.44 0.44 0.44 0.44 5.5 1.25 0.94 0.10 0.08 0.47 0.50 0.47 0.44 6.5 1.00 0.81 0.01 0.00 0.40 0.40 0.40 0.41 7.5 1.00 0.50 0.19 0.09 0.25 0.25 0.25 0.26 8.5 2.00 0.36 0.19 0.09 0.18 0.18 0.18 0.18 "1.0M NH4HSO•-3.0M urea, 32 ø C, 50:1 bath ratio. b %MNP•. • MNP• + ¾•(MNP•-MNP•). a MNP•-[MNP• + ¾•(MNP•-MNP•)]. The first analysis was performed on the bisulfite-treated hair where the CySSOa- to CySH ratio would be expected to be one. These results are summarized in Table VII for hair treated with bisulfite at pH's ranging from 3.5 to 8.5. The data clearly show that the CySSOa- to CySH ratio for hair treated in this broad pH range is one. These results are totally consistent with the general cystine-bisulfite reaction mechanism and reflect the pH dependence of the cystine-bisulfite equilibrium (8). It also shows that the analytical method is applicable to hair treated with bisulfite at higher pH's than previously observed. The method was next applied to hair containing excess cysteinyl residues. The results are described in Table VIII. For hair treated with thioglycolate followed by bisulfite, there is obtained a total CySSOa- content of 0.17 meq/g and a CySH content of 0.34 meq/g. For hair containing excess CySSOa- residues, the treatment of hair with bisulfite-tetrathionate followed by bisulfite was considered. The data are