The reduction of iodonitrotetrazolium chloride by ferredoxin-NADP + reductase: A new tool for the characterization of the spinach chloroplast flavoprotein

Abstract

A thorough investigation on the long-known reactivity of spinach ferredoxin-NADP + reductase with tetrazolium salts has allowed the setting-up of a new assay, highly specific for this enzyme. By immunological titration it has been determined that more than 92% of the iodonitrotetrazolium chloride reductase activity of spinach leaves' homogenate belongs to ferrodoxin-NADP + reductase. It has been shown that addition of formazanbinding agents, like Triton X-100, is required for the assay of the partially purified enzyme; furthermore Triton X-100 enhanced considerably the tetrazolium salts reductase activity of all the enzyme preparations tested. The optimal condition of pH, ionic strength and Triton X-100 concentration have been determined. Kinetic experiments yielded a series of parallel lines in the double-reciprocal plots and the kinetic parameters obtained are reported. Moreover it has been shown that at least 75–80% of the activity with tetrazolium salts of ferrodoxin-NADP + reductase is not mediated by the superoxide radical.