STUDIES ON THE ENZYMATIC BASIS FOR THE JANUS GREEN B STAINING REACTION

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Abstract
Enzymatic reduction of Janus green B by isolated enzyme systems was studied with the Warburg respirometer. Degree of dye reduction was evaluated by color change or spectrophotometrically. Various components of the dye system (Janus green B, leuco Janus green B, diethylsafranine, and leucosafranine) were detd. spectrophotometrically, by absorption spectra maxima, or extinction at different wave lengths. Janus green B was found to be reduced by reduced flavoprotein, hence the dye can be reduced by any enzyme system capable of reducing DPN and thereby reducing flavoprotein. Selective staining of mitochondria within the intact cell was attributed to a more rapid rate of reduction of Janus green B by non-mitochon-drial portions of cell; slower reduction rate in mitochondria was attributed to concn. of cytochrome oxidase system within the organelles. Preferential adsorption was not found to play a primary role in staining of mitochondria. Although mitochondria stain selectively with Janus green B in the intact cell, the staining of isolated structures with the dye does not necessarily prove the structures to be mitochondria. Mitochondria, microsome, and supernatant fractions from liver cell homogenates were all found to be capable of reducing the dye under appropriate conditions.