Histochemical Localization in Animal Tissues of a Special Peroxidase which Generates the Free Radical of Ascorbic Acid

Abstract

For the histochemical localization of a special ascorbic acid-free radical (AA.FR) peroxidase, frozen sections (10–15 _μ) of fresh liver and striped muscle from pigeon and domestic fowl were immersed for 18–24 hr in an incubation medium consisting of: saturated aqueous o-dinitrobenzene, 5 ml; ascorbic acid 5 mg/ml, 5 ml: 3% H₂O₂, 1 ml; and citrate buffer pH 6.4–6.6, 2 ml. Sections were then rinsed with water, treated with 1% NH₄OH for 2 min, rinsed again with water and mounted in glycerol jelly. The cells containing the AA-FR peroxidase acquired a deep purple color. Three controls consisted of: (i) replacement of AA with 5 ml of water; (ii) addition to the incubating medium of 5 ml of 0.002 M Na₂S₂O₅; or (iii) addition of 2 × 10⁻²M catalase (from buffalo Liver—Biochemical Unit, V. P. Chest Instt. Delhi-7). A higher peroxidase activity was observed in the liver than in the muscle. Among the red and white fibres of muscle, the former were comparatively richer in the enzyme, which appeared to be mitochondrial in localization. A higher peroxidase activity is correlated with active metabolism of tissues such as the liver and the fat-utilizing red muscle fibres.