Factors Affecting the Ellman Determination of Sulfhydryl Groups in Skimmilk Powder and Gels

The stability of thionitrophenylate, the product of Ellman's reaction with sulfhydryl groups, increased over the pH range 7 to 9 when the solutions were kept at 4C. At room temperature, loss of yellow coloration at pH 8 was prevented with ascorbic acid. Maximum exposure of the masked sulfhydryl groups of skimmilk powder occurred with 3M guanidine hydrochloride, but greater absorbance stability was achieved in 5M urea. Sodium lauryl sulfate acted slowly in exposing masked sulfhydryl groups. Sodium borohydride as a reducing agent and a mixture of acetone and hydrochloric acid to destroy excess reductant made Ellman's procedure adaptable to the estimation of disulfide. On thermal gelation of skimmilk, a maximum increase (55%) of free sulfhydryl groups occurred after five minutes in gels heated at 100C and after one minute in gels heated at 115C. In spite of changes in free sulfhydryl group titer, the total of free and masked sulfhydryl and disulfide groups remained constant during the heating periods.