Studies on the phenazine methosulphate-tetrazolium capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. II. A novel hypothesis for the mode of action of PMS and a study of the properties of reduced PMS

Abstract

The results in the preceding paper have shown that the PMS-tetrazolium capture reaction as such is not sufficient to guarantee a correct localization of formazan in microscopically small dehydrogenase sites. For cytochemical reactions where the application of PMS leads to increased formazan formation, it is proposed that PMS functions not on its own, but as an efficient acceptor of NAD(P)H-oxidizing flavoproteins and thus increases the local NAD(P)H tetrazolium oxidoreductase activity. For the redox mediator vitamin K3 this type of mechanism could be proven with rat liver fractions. The relatively rapid NADPH oxidation precluded such simple experiments with PMS. An indication of such a stimulation by PMS was, however, obtained with soluble rat liver fraction. As escape of reducing equivalents from the site might also occur at the level of reduced PMS (PMSH) the solubility properties of PMSH were studied. It was found that PMSH has a low solubility in aqueous media and is hydrophobic. On basis of these findings a 'post-tetrazolium reduction' method seemed possible and could be experimentally confirmed.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate

“…The hydrophilic cation PMS is now widely used as an intermediate redox reagent for coupling reduced dehydrogenase coenzymes to the reduction of tetrazolium salts into colored formazans [7,8,9,10,11,12,13,14]. A similar electron-transfer agent, 1-methoxy-PMS, has also been used for revealing dehydrogenase activity in living hepatocytes [15].…”

“…The reduced PMS (methyl-phenazine, MPH) is colorless and easily reoxidized by oxygen or via the respiratory chain, and the reaction is blocked by inhibitors of cytochrome oxidase such as cyanide ions [22]. Methyl-phenazine is hydrophobic and has low solubility in aqueous media [12]. The chemical structures of PMS and methyl-phenazine are shown in Figure 1.…”