On the Determination of Cystine as Cysteic Acid
نویسنده
چکیده
In the procedure of Schram, Moore, and Bigwood (1) for the determination of the cystine plus cysteine content of a protein, the directions for the concentration of the reaction mixture need to be followed explicitly in order to avoid overoxidation. The addition of a reducing agent to destroy the excess performic acid before the concentration step would seem appropriate if the reductant would have no deleterious effect on the subsequent acid hydrolysis or the chromatographic determination of cysteic acid. We have tested a number of reducing agents for this purpose. The most generally useful results have been obtained with HBr. The bromine formed is volatile and can be rapidly removed from the reaction mixture under reduced pressure. Bromine itself is an effective reagent for oxidizing cystine to cysteic acid, yet Thompson (2) found that with proteins he could not obtain high yields of cysteic acid by bromine oxidation. The data indicated, however, that if performic acid were used for the initial reaction, the subsequent brief exposure of cysteic acid residues to bromine would not be likely to be detrimental. The present experiments started from this premise. Methionine sulfone is more sensitive to overoxidation by performic acid than is cysteic acid.1 The yield of methionine sulfone can be improved by removing the performic acid by lyophilization rather than by rotary evaporation. The use of HBr, however, has invariably permitted quantitative yields of methionine sulfone. The present procedure thus provides a method for determining both cystine plus cysteine and methionine as their oxidation products. The determination, as was the earlier one (l), is also fully applicable to carbohydratecontaining samples.
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