LDH ISOENZYMES AS CAUSE OF UNSPECIFIC TETRAZOLIUM SALT STAINING IN GEL ENZYMOGRAMS (NOTHING DEHYDROGENASE)

It was demonstrated that after tetrazolium salt staining of gel enzymograms of extracts containing NAD-dependent dehydrogenases, a pattern of lactate dehydrogenase (l-lactate: NAD-oxidoreductase, E.G. 1.1.1.27) isoenzymes also became visible, although the incubation mixture contained no lactate, but only the specific substrate for the dehydrogenase examined. From the experiments reported it appears that the supporting media of the gel electrophoresis (starch, polyacrylamide, cellulose acetate) contain groups similar to lactate insolubly bound to the polymerous gel system. This is probably the case also with agar gel. This fault unavoidably occurs with all methods used. By differential staining of horizontal layers of a gel with different substrates it was shown that extracts of human tissue contained only a single anodic band of glycerol-3-phosphate dehydrogenase (l-glycerol-3-phosphate: NAD-oxidoreductase, E.G. 1.1.1.8) and a single anodic band of glutamate dehydrogenase (l-glutamate: NAD-oxidoreductase, deaminating, E.G. 1.4.1.3). The remaining bands that could be visualized by usual staining methods have been identified as lactate dehydrogenase isoenzymes. Confirmation was obtained by comparison with results of ion exchange Chromatography. These results offer a simple explanation for the unspecific staining of lactate dehydrogenase isoenzymes, and make it possible to avoid erroneous conclusions in the examination of multiple forms of enzymes, as seen in numerous reports recently. © 1968.