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Determination of Sulfhydryl and Disulfide Groups in Milk by the p-Chloromercuribenzoate-Dithizone Method¹

Department of Food Science, University of Illinois, Urbana

ABSTRACT

Estimations reported for -SH and -SS- in milk have differed because of variations in methodology and milk composition. For these reasons the p-chloromercuribenzoate-dithizone (PCMB-D) method of Fridovich and Handler (Anal. Chem., 29:1219. 1957.) was adapted to milk. This method is simple, rapid, of high reproducibility, and yielded 2 M -SH and 3 M -SS- per mol wt of 35,000 for ß-lactoglobulin. The values for raw milk were 0.16 ± 0.02 and 0.97 ± 0.06, and for sterile concentrated milk they were 0.27 ± 0.012 and 2.69 ± 0.05 mM per liter of -SH and -SS-, respectively. Maximal -SH and -SS- were obtained simultaneously with 6-8 M urea; whereas, 0.25% sodium lauryl sulfate increased -SH, but larger amounts decreased it. When cysteine and cystine were added to milk, their recoveries were 94.4 ± 1.1% and 98.3 ± 4.6%, respectively. A plot of PCMB versus decrease in OD at 620 mµ, was linear up to 11 mM per liter of PCMB. Absorbence for the PCMB-D reaction with and without urea revealed no significant differences within 350 to 750 mµ.

¹ This research was supported by the U. S. Public Health Grant Ef-141.

² Postdoctorate Research Associate.