CYSTEINYL AND S-SULFOCYSTEINYL BESIDUES 463 trolled, blocking of the Ker-CySH prior to keratin hydrolysis. Nitroprusside is used as the indicator in the reaction. This mercurial-nitroprusside titration procedure is abbreviated as MNP. Derivation of the Method The new method reported here is essentially a variation of that of Valk and Gerthsen but with some changes introduced to avoid losing CySSOa- residues when the pH is raised in an uncontrolled manner. Additionally, the alkylation of CySH residues is carried out in a more quantitative fashion. For this new method, three separate subsamples are required for the simul- taneous determination of CySH and CvSqO.:- Subsample 1 is hydrolyzed in acid and the cysteinyl content is determined by mercurial titration. This step, which is identical to Valk and Gerthsen's initial step, yields the MNP• value and is a measure of the total cleavage level of the keratin sample assuming that the CySSCy is cleaved in a nucleo- philic process (e.g., by bisulfite, thioglyeolate, ete). 6N H2SO,t x Ker-CySH + y Ker-CySSOa- • (x + y) CySH Subsample 2 is extensively water-rinsed and treated with alkali to reverse the cystine-bisulfite reaction in a controlled manner. The sample is hydro- lyzed in acid and the results obtained are expressed as the MNP2 value. x Ker-CySH + y Ker-CySSOa- (OH-) • z Ker-CySSCy-Ker + (x - z) Ker-CySH + (y - z) Ker-GySSOs- 6N HeSO4 • (x+y-2z) CySH Clearly, one-half of the value of MNP• minus MNPo is a measure of the amount of CySSOa- residues consumed during the reversal step. Subsample 3 is water-rinsed and treated with alkali in a manner identical to subsample 2, and is then alkylated with acrylonitrile. This treatment con- verts remaining CySH groups to nontitratable fi-cyanoethylsulfide residues (CySCH2CH2CN). Because the alkylation is performed after alkali reversal, no further reversal during alkylation is likely. The sample is then hydrolyzed in acid and the MNP.• titer is determined. (On-) x Ker-CySH + y Ker-CySSOa- , z Ker-CySSCy-Ker + (x - z) Ker-CySH + (y - z) Ker-CySSOa-CH• = CHCN

464 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS z Ker-CySSCy-Ker + (x -. z) Ker-CySCH2CH2CN + (y -- z) Ker-CySSO 4- 6N H2SO4 • (y-z) CySH This titer is then a measure of the CySSO•- content which has survived the reversal reaction. The MNP1, MNP2, and MNP:• determinations are summarized below. MNP• = sum of combined Ker-CySH and Ker-CySSO:•- MNPa + 1/2 (MNP1 - MNPQ = Ker-CySS0a- content of original sample MNP• - [MNPa + 1/2 (MNP1 - MNP,2)] = Ker-CySH content of original sample MNP1 is the sum of the combined CySH and CySSO:•- content. The CySSOa- content of the original sample is then the MNPa titer, that is, the CySSOa- content which survived reversal and alkylation, plus one-half tim value of MNP1 minus MNP2, the CySSOa- content of the original sample. The CySH content of the original sample is obviously then MNP1 minus the CySSOa- content of the original sample. EXPERIMENTAL Materials Brown, European, human hair* was cleaned by treatment with an aqueous solution of anionic detergent and then used throughout this investigation. The analytical results are expressed on the basis of vacuum oven-dried (one hour, 105øC, ~1 mm Hg pressure) hair weight. All reagents employed were the best grade available and were used with- out further purification. Salyrganic acidP (Fig. 1) was used as the organic mercurial titrant. The mercurial was made up to approximately 3 x 10 -a mo- lar concentration in 3 x 10 -2 molar aqueous sodium chloride. Klotz and Carv- * DeMeo Brothers, New York, N.Y. 'i' Winthrop Laboratories, New York, N.Y. • OCH.•CO.•H CNHCH.,CHCH.,HgOH 0 OCH• Figure 1. Salyrganic acid