Studies on the Peroxidase Effect of Cytochrome c. I. Peroxidase Activity in Subcellular Fractions from the Rat Kidney, and the Assay of Soluble Cytochrome c by Paper Electrophoresis.

Abstract

Homogenates of perfused rat kidneys have been prepared in isotonic sucrose and in saline, and their subcellular fractions examined for peroxidase activity. Using a benzidine-peroxide-nitroprusside solution as screening reagent for peroxidase activity, the following results were obtained A positive peroxidase reaction was demonstrated in all subcellular fractions. The peroxidatically active components of the soluble fraction were studied electrophoretically with paper or starch-gel as the supporting medium. The main peroxidase activity of the soluble fraction could be attributed to cytochrome c. Paper electrophoresis provided a simple and reliable routine method for the detection and assay of small amounts of soluble cytochrome c in tissue homogenates and extracts. As little as 3 x 10-2 [mu]g cytochrome c was clearly visible by means of the peroxidase staining. Starch-gel electrophoresis constitutes a less sensitive method of detecting cyto-chrome c than paper electrophoresis. The peroxidase activities in the mitochondrial and the submicroscopic particle fractions are discussed.