On the role of oxygen in dehydrogenase reactions using tetrazolium salts.

Abstract

Fundamental aspects of the reduction of tetrazolium salts were investigated and, in particular, the role of oxygen in the reduction. It was found that oxygen had a competitive inhibitory effect on the reduction of (Tetra)Nitro BT mediated by NADH and phenazine methosulphate. This competitive effect, under aerobic conditions, could be reversed by using tetrazolium concentrations of 5 mM. Oxygen did not have a significant effect on BPST reduction, whereas the inhibitory effect of oxygen on the reduction of Neotetrazolium was not reversed by increasing the tetrazolium concentration. The oxygen effect on Nitro BT reduction was considerably less when macromolecular substances such as albumin or polyvinyl alcohol were added to the medium. This may be due to increased Nitro BT concentrations being built up at the surface of macromolecules due to the nonpolar components of the Nitro BT molecule. When demonstrating glucose-6-phosphate dehydrogenase activity in vitro or in tissue sections with the use of Nitro BT, oxygen also had a direct inhibitory effect, even when azide was added to the medium for the inhibition of flavoprotein-mediated electron transfer to oxygen. Again, this direct inhibition of Nitro BT reduction by oxygen could be excluded by using a high Nitro BT concentration. Macromolecules present in the incubation medium or in tissue sections counteracted the oxygen effect. It is concluded that the maximum reaction rate and optimum localization of dehydrogenases is obtained when histochemical media are used containing 5 mM (Tetra)Nitro BT and 20% polyvinyl alcohol.