AQUEOUS SOLUBLE TETRAZOLIUM FORMAZAN MTS AS AN INDICATOR OF NADH-DEPENDENT AND NADPH-DEPENDENT DEHYDROGENASE-ACTIVITY/

Recently a new tetrazolium was described for the use of monitoring cel l viability in culture. This tetrazolium, commonly referred to as MTS 5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol) -2-(4-sulfophenyl)- 2H-retrazolium, inner salt], has the unusual property that it can be r educed to a water-soluble formazan. beta-Nicotinamide adenine dinucleo tide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phospha te/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduced to the soluble formazan in the presence of NADH and NADPH, and reactions were compared to thos e with dithiothreitol (DTT) or 2-mercaproethanol (2-ME). The efficienc y of these reactions was enhanced 1000-fold by the presence of phenazi ne methosulfate. Selectivity in the electron transfer from NADPH was s lightly 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,,vas m onitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of the NADH or NADPH. This system rep resents a useful tool for evaluating reaction kinetics in solutions of NAD- and NADP-dependent dehydrogenase enzymes, and these reactions ca n be performed in typical biological buffers containing reducing agent s without significant interference to the MTS/formazan system.