J. Soc. Cosine*. Chem., 25, 461-472 (September 1974) The Simultaneous Determination of Cysteinyl and S-$ulfocysteinyl Residues in Keratin PHILLIP E. SOKOL, Ph.D.,* FRANCIS H. GIRARD, Ph.D.,* D. Y. HSIUNG, Ph.D.,* and CAROLYN PICTOR, B.S.* Presented October 9, 1973, Joint Symposium o[ the Society o[ Cosmetic Chemists and the Association ol r O•cial Analytical Chemists, Washington, D.C Synopsis-An analytical method is described for the simultaneous determination of S-SUL- FOCYSTEINYL (CySSOa-) and CYSTEINYL (CySH) residues in KERATIN. This nexv method, a modification of Valk and Gerthsen's procedure, consists of the SALYRGANIC ACID MERCURIAL TITRATION of acid hydrolysates of treated keratin in which CySH residues are differentiated from CySSO•- residues by a simple, but controlled, blocking of the CySH with acrylonitrile prior to keratin HYDROLYSIS. Publication of this method was, in part, stimulated by Valk and Gerthsen's report that the CySH/CySSO•- ratio in sulfite-treated keratin is dependent upon the pH of the treat- ment medium, deviating largely from 1.0 outside the pH range 3-6. The method reported here indicates that the stoichiometry of the reaction Ker-CySSCy-Ker q-HSO•-• Ker- CySH q- Ker-CySSOa- is obeyed at all pH's examined (3.5-8.5). The method is precise, accurate, and rapid. In addition, the method is valid for keratin samples containing a wide range of CySH/CySSOa- ratios as well as samples containing one of these groups ex- clusively. INTRODUCTION For a number of years xve have been interested in the chemistry of the keratin-bisulfite reaction. The reaction, Ker-CySSCy-Ker + HSOa- --• Ker- CySH + Ker-CySSOa-,-* is the basis of several commercial products. We were interested in determining the exact extent of this reaction as we]] as Gillette Research Institute, Rockville, Md. 20850. -• Gillette Co. Personal Care Division, Boston, Mass. 02106. :t: Ker- represents the noncysteinyl and noncystiny] containing portions of the keratin pro- tein. 461

462 JOUBNAL OF THE SOCIETY OF COSMETIC CHEMISTS in determining the relative amounts of cysteinyl (CySH) and S-sulfoeysteinyl (CySSO3-) residues produced in the reaction since, under certain reaction conditions, one might not be expected to obtain equal amounts of each group. The objective of this work was, therefore, to develop an analytical method which quantitatively determines the amounts of CySSOa- Bunte salt and CySH thiol groups in chemically modified keratin. The analytical determination of these residues presents several difficult problems. For example, the method of Elsworth and Phillips (1), which measures the sulfur dioxide evolution after treatment of the modified keratin sample with add, is very laborious and requires replicate samples which contain identical amounts of sorbed bisulfite. Further, the procedure deter- mines only CySSOa- residues and cannot be used to detcrmine CyStt resi- dues. The iodoacetamide procedure (2) and polarographic methods (3) are not useful for the determination of both CySSOa- and CySH residues be- cause at the high pH's needed, reversal of the CySSCy + HSO:4- reaction occurs rapidly and leads to false results. To overcome these difficulties, Valk and Gerthsen (4) expanded the mer- curial titration procedure for CySH residues to determine CySSOa- residues also according to the following scheme: HSO.•- Ker-CySSCy-Ker • a Ker-CySH + b Ker-CySSOa- a Ker-CySH + b Ker-CySSOs- H2SO4 -(a + b) CySH + b SO4 = ICHaCOsH H2504 a Ker-CySH + b Ker-CySSOa- -• (On •-) a a CySCH2CO2H 4- b CySH + b SO4: (3) From eq 2 the combined CySH and CySSOa- contents are determined. From eq 3 the CySH residue is blocked with iodoacetate and, therefore, the CySH content resulting from the acid hydrolysis of the CySSOa- groups is deter- mined. Using tl•is procedure, Valk and Gerthsen found CySSOa-/CySH ratios of unity for wool reduced with bisulfite over the pH range from 3 to 6. Outside of this pH range, the ratios were significantly different from unity which, they suggested, was due to a change in the keratin-bisulfite reaction mechanism. We had reason to doubt this was the case and were able to show the ratio to be unity at all pH's studied-from 3.5 to 8.5. We also had need for an independent determination of CySH and CySSO:•- residues in keratin fibers for product development studies. The procedure developed for this determination consists of the mercurial titration of acid hydrolysates of chemically treated keratin in which CySH residues are differentiated from CySSOa- residues by a simple, but con-