CYSTEINYL AND S-SULFOCYSTEINYL RESIDUES 471 Table VIII Analysis of Hair Treated •vith Thioglycolate (TGA)" Followed by Bisulfite b meq SH/g MNP• of TGA treatment 0.19 MNP (after TGA and bisulfite treatment) MNPi 0.53 MNP., 0.26 MNP., 0.06 Ker-CySSO3- (calculated •) 0.17 Ker-CySSO3- (found) 0.19 Ker-CySH (found a) 0.35 • 1.0-hour immersion in 0.15N ammonium thioglycolate, pH 9.2, 32øC, 50:1 bath ratio, 30-min •vater rinse under N, b 15-rain immersion in 0.5M NH4HSO3-1.0M urea, pH 6.5, 32øC, 50:1 bath ratio. • Ker_CySSO•- (calculated): [MNP•-MNP• øf TGA treatment 1 2 ' a Ker-CySH (found) = [MNP• (after TGA and bisulfite treatment)-Ker-CySSO,- (found)]. Table IX Analysis of Hair Treated •vith Bisulfite-Tetrathionate • Followed by Bisulfite • meq SH/g 0.26 MNP• of bisuhqte-tetrathionate treatment MNP (after bisulfite-tetrathionate treat- ment and bisulfite treatment) MNPi MNP, MNP, Ker-CySSO•- (calculated *) Ker-CySSO.•- (found) Ker-CySH (found a) 0.61 0.32 0.26 0.44 0.40 0.21 10-min immersion in 1.0M NH•HSO•-4.0M urea-4.0M tetrathionate, pH 8.0, 50:1 bath ratio, 32 ø C, followed by 30-min immersion in water. 15-min immersion in 0.5M NH•HSO•-i.5M urea, pH 6.5, 50:1 bath ratio, 32øC. Ker-CySSO•- (calculated) = ¾2 [MNP• + MNP• of bisulfite-tetrathionate]. Ker-CySH (found) = [MNP•-Ker-CySSO.•- (found)]. reported in Table IX. Again there is seen consistent behavior. The CySSOa- content is 0.44 meq/g while the CySH content was found to be 0.21 meq/g. For hair contai•fing S-sulfocysteinyl residues only as previously discussed (Table V), evidence of only CySSOa- residues is observed no CySH resi- dues can be determined. Similarly, for hair treated with thioglycolate only (Table IV) cysteinyl residues are observed.

472 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS A final point to be made in these studies is the very small amount of cysteine lost, less than 0.05 meq, when either reduced or untreated hair is hydrolyzed in acid in the presence or absence of added cysteine. In the absence of hair, cysteine can be recovered from the hydrolysis and titration procedure in quantitative amounts. In the presence of hair, however, the very small amount, 0.05 meq, is lost. The reason for this loss is not •vell understood but the magnitude of the loss clearly does not interfere xvith the utility of the procedure. CONCLUSIONS A method has been developed for the quantitative differentiation of CySSO,q- and CySH residues in chemically treated keratin. The procedure was validated by verifying the assumptions on which it is based and showing that keratin, in this ease human hair, containing predictable CySSOa- to CySH ratios gave analytical results consistent with expectations. The method was shown to be applicable to analyzing keratin treated under a variety of conditions including acidic and alkaline bisulfite. The method was also shown to be useful on hair containing a wide range of CySSOa- to CySH ratios as xvell as hair containing only CySSOa- or CvSH groups. (Received January 31, 1974) REFERENCES (1) Elsworth, F. F. and Phillips, H., The examination of "sulfur stored" and "sulfited'" wool fabrics for sulfur dioxide and sulfite, J. Tex. Inst., 29, T219 (1938). (9.) Stein, H. H., an•d Guarnaccio, J., The determination of sulfhydryl groups in reduced hair keratin, Anal. Chim. Acta, 23, 89 (1960). (3) Leach, S. J., The reaction of thiol and disulfide groups with mereuric chloride and methylmercuric iodide. I. Simple thiols and soluble proteins, Aust. J. Chem., 13, 520 (1960). (4) Valk, G., and Gerthsen, T., A possible method for determination of groups of colored salts attached to wool, Melliand Texti•ber., 47, 407 (1966). (5) Klotz, I. M., and Carver, B. B., A spectrophotometric titration for the determination of suli•hydryl groups, Arch. Biochem. Bioph•!s., 95, 540 (1961). (6) Wolfram, L. J., private communication, 196•. (7) Menkart, J., and Wolfram, L. J., U.S. Patent 3,•67,363 (March 2, 1971). (8) Wolfram, L. J., and Underwood, D. L., The equilibrium between the disu]fide linkage in hair keratin and sulfite or mereaptan, Text. Res. l., 36, 947 (19Ff).