Superoxide anion radical-independent pathway for reduction of tetrazolium salts in aerobic mixtures consisting of NADH and 5-methylphenazinium methyl sulfate in the presence of aqueous micelles of nonionic and cationic detergents

Abstract

The effect of Triton X-100 and certain other nonionic as well as cationic detergents on 5-methyl-phenazinium methyl sulfate (PMS)-mediated reduction tetrazolium salts was studied under aerobic conditions using an exogenous source of reducing equivalents, such as NADH or by generating NADPH through an enzymatic reaction. In the absence of detergents, 5,10-dihydor-5-methylphenazine (MPH), formed on reduction of 5-methyl-phenazinium cation (MP +) of PMS by NAD[P]H, was reoxidized allowing first the univalent reduction of molecular oxygen (O 2) to the superoxide anion radical O 2 ⪰ which turn, reduced tetrazolium salts. In the presence of detergents, however, a significant fraction of the PMS-mediated reduction of tetrazolium salts appeared to proceed without the intervention of O 2 ⪰. The reasons for this were examined experimentally and it was suggested that the reduced phenazine (i.e., MPH), which is sparingly soluble in aqueous solutions, migrates into detergent micelles where tetrazolium salts are reduced in preference to O 2. By lowering the pH and thereby facilitating the H +-mediated dismutation of O 2 ⪰, it was possible to obtain the reduction of tetrazolium salts, mediated selectively and directly by MPH in the micellar pseudophase. Employing the technique of saturation analysis, further evidence was obtained that lends support for preferential reduction of tetrazolium salts (e.g., nitroblue tetrazolium chloride) to that of O 2 by the micelle-bound MPH.