STUDIES OF INDOPHENOL BLUE SYNTHESIS: I. THE ROLE OF FREE RADICAL FORMATION BY HEART MUSCLE PARTICULATES DURING THE "G" NADI REACTION

A study of the "G" Nadi reaction mediated by Keilin and Hartree beef heart muscle preparations has revealed that of the two Nadi reagents, i.e., dimethylparaphenylenediamine and α-naphthol, only the diamine is enzymatically oxidized by the terminal respiratory enzymes of the heart particulates. In both spectrophotometric and manometric experiments, enzymatic oxidation of α-naphthol by the heart muscle enzymes could not be detected. Oxidation of the diamine resulted in formation of a stable free radical and polymers with characteristic absorption spectra. The free radical and polymers, upon formation, rapidly reacted with the α-naphthol in a non-enzymatic oxidative coupling to form indophenol blue. Incubation of dimethylparaphenylenediamine alone with heart muscle particles resulted in production of an insoluble blue dye in the particles. Upon extraction with 95% ethanol, the pigment, which is not indophenol blue, exhibited a broad absorption maximum at 460-470 mµ.