A high-sensitivity, single-gel, polyacrylamide gel electrophoresis method for the quantitative determination of glutathione reductases.

A new method is described that allows the selective staining and quantification of gluthathione reductases (EC 1.6.4.2) in cell extracts following acrylamide gel electrophoresis. The method is based on modifications of two previous procedures; it uses DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] to develop a yellow color on reaction with GSH formed from the NADPH-dependent reduction of oxidized glutathione. This is followed by specific counterstaining of glutathione reductase with dichlorophenolindophenol/nitroblue tetrazolium. The use of DTNB in the initial staining step inhibits enzymes other than glutathione reductase that could be stained with the dichlorophenolindophenol/nitroblue tetrazolium counterstain. Enzymes such as thioredoxin reductase, which can directly reduce DTNB with NADPH, may be nonselectively stained by this new procedure. Plant ferredoxin-thioredoxin reductase is not reduced by NADPH and therefore does not appear. Glutathione reductase stains much quicker with DTNB in the presence of GSSG than with thioredoxin reductase, allowing them to be distinguished, if parallel gels are run without GSSG, where the two enzymes react at the same rate. The sequential use of two staining procedures results in distinct, sharp permanent bands that can be used to quantify the activity of glutathione reductase while precluding artifacts generated by the previous methods.