Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine

The total glutathione content of biological samples is conveniently determined with an enzymatic recycling assay based on glutathione reductase (F. Tietze, 1969, Anal. Biochem.27, 502–522). In the original and several subsequent descriptions of this procedure, glutathione disulfide is selectively determined by assaying samples in which glutathione is masked by pretreatment with N-ethylmaleimide. Since N-ethylmaleimide is a potent inhibitor of glutathione reductase, it is necessary to remove excess reagent; the procedures used are laborious and contribute significantly to experimental error. It is reported here that 2-vinylpyridine is a much better reagent for the derivitization of glutathione. In contrast to N-ethylmaleimide, 2-vinylpyridine does not inhibit glutathione reductase significantly and therefore need not be removed from the sample solutions. 2-Vinylpyridine reacts with glutathione at slightly acidic pH values where spontaneous formation of glutathione disulfide is minimal. It is demonstrated that the total glutathione concentration in mouse plasma is substantially higher than generally reported and that glutathione disulfide constitutes less than 30% of the total glutathione present.