Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.
Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.
Dunigan
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DD
Department of Biology, University of South Florida, Tampa 33620-5150, USA.
Waters
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SB
Owen
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TC
D004229:Dithiothreitol / Q000494:pharmacology; D005562:Formazans / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008773:Methylphenazonium Methosulfate / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009245:NADH Dehydrogenase / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*
D005562:Formazans; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009243:NAD; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D008773:Methylphenazonium Methosulfate; D009249:NADP; D008623:Mercaptoethanol; D009252:NADPH Dehydrogenase; D009245:NADH Dehydrogenase; D004229:Dithiothreitol