An Ultrasensitive Assay for Soluble Sulfhydryl and Its Application to the Study of Glutathione Levels during the Hela Life Cycle

Changing glutathione levels have often been implicated in mitotic events since Rapkine discovered the cyclic variation of a trichloroacetic acid (TCA)-soluble sulfhydryl (SH)-containing molecule in synchronously dividing sea urchin embryos (1) . However, Waley has pointed out that many studies of glutathione fail to use assays of sufficient specificity, in that small peptides, ascorbate, and cysteine frequently lead to ambiguous measurements (2) ; indeed, reexamination of the Rapkine cycle, at least for sea urchin, has disclosed that the variation in TCA-soluble SH content is due to a small peptide other than glutathione (3) . In order to examine glutathione levels in a mammalian cell during its life cycle, we have developed a simple, specific, and comparatively sensitive procedure (see 4 for review) applicable to samples as small as 0.5 mg wet weight . Briefly, the 100,000 g supernate from a cell lysate is reacted with N-ethylmaleimide14C (NEM) . The reaction with SH involves addition of a sulfanion to the double bond of the imide in NEM and is specific for the SH grouping at pH 7 over a 15 min interval (5) . The glutathione adduct is then isolated from other SH-containing (radioactive) species by acrylamide gel electrophoresis and quantitated by scintillation counting. States and Segal have also used NEM14C to separate the glutathione adduct from a complex biological system on thin-layer chromatography ; however, no attempt at quantification was made (6) .