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

CYSTEINYL AND S-SULFOCYSTEINYL RESIDUES 465 er (5) used this mercurial previously and had found it to be more soluble than many of the other mercurials. The salyrganie add solution was stan- dardized against a sample of eysteine of known purity. A 1% aqueous solu- tion of sodiron nitroprusside was used as the indicator for the mereurial- eysteine reaction, the color change at the endpoint being pink to colorless. Methods Each analysis requires three preweighed subsamples of approximately 0.1 to 0.2 g taken from the treated keratin specimen to be analyzed. Where not spedfled, liquor-to-keratin ratios in the analytical procedure are 125:1 or greater. After treatment, each keratin sample is stripped of excess treat- ment liquor by blotting with filter paper and then washed by immersion in three portions of 6N H2SO4. Subsample i is hydrolyzed for MNP1 titer. MNP hydrolyses are earfled out in about 85 ml of 6N sulfurie add, for 17 hours at 95øC. The effects of varying the time and temperatures of hydrolysis were briefly studied. Com- parable results could be obtained with shorter hydrolysis times at higher temperatures in an evacuated bomb, for example 3•/• hours at 160øC. We prefer, however, the hydrolysis at 95øC because of its relative safety and simplicity. In all eases we found that the hydrolysate contains a small amount of fibrillar material, less than 3% by weight of the hair sample, which does not appear to interfere with the analysis. Subsample 2 is blotted with filter paper and washed by immersion in two portions of deaerated distilled water under nitrogen to prevent oxidation of sulfhydryl groups. The sample is blotted and alkali-reversed by successive immersion in two portions ooe 0.2M sodium sesquiearbonate under nitrogen. The sample is next washed by a 1-min immersion in the deaerated distilled water under nitrogen and then hydrolyzed to obtain the MNP2 value. Subsample 3 is treated as was subsample 2 prior to the hydrolysis step. The sample is then blotted and immersed in a 5% solution of aerylonitrile in 0.1M, pH 9.2 borate buffer at 32øC for 30 min. The sample is then rinsed in running water for I rain and then hydrolyzed to obtain the MNP.• titer. After hydrolysis, the samples are cooled to room temperature and diluted to 100 ml with distilled water. It was found convenient to carry out the hydrolysis in a 100-ml volumetric flask. A 5- or 10-ml aliquot is removed and added rapidly with stirring to four times its volume of saturated sodium carbonate solution. This produces a blanket of carbon dioxide which in- hibits aerial oxidation of mercaptide. Salyrganic acid solution is added from a buret, and 10-15 drops of nitroprusside are added just before the end- point. One or two titrations are necessary to determine the approximate endpoint. Care should be taken to avoid early addition of nitroprusside since it decomposes to an orange-colored compound in the alkaline medium which obscures the endpoint.