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Cytosolic functions obtained from various bovine tissues was individually subjected to column isoelectric focusing in order to resolve the glutathione S-transferase isoenzymes. The results showed a large variability in the isoenzyme pattern. All the tissues were found to have neutral-acidic forms of the enzyme, whilst liver, adrenal gland, testicle, lung and kidney contained a conspicuous amount of activity associated with the cationic forms of the enzyme. In spite of these differences, by comparison of the conjugating activity of transferases, we did not find essential inter-organ variations. Conversely, when the same tissue samples were tested for selenium independent glutathione peroxidase activity, using cumene hydroperoxide as second substrate, we observed a higher activity in the organs having the cationic form of glutathione S-transferase.

Exposure of purified human hemoglobin to phenylhydrazine induces the oxidation of hemoglobin and the generation of acid soluble peptides. The extent of protein fragmentation depends on the concentration of phenylhydrazine, incubation time and temperature. The fragments, excluded by gel filtration chromatography on Sephadex G-15, are partially degraded by leucine aminopeptidase and are totally converted to amino acids by acid hydrolysis. The addition of inhibitors for serine proteinases (phenylmethylsulfonylfluoride), cysteine proteinases (leupeptin), aspartic proteinases (pepstatin A) and metalloproteinases (EDTA) does not alter the formation of acid soluble peptides, thus excluding the involvement of erythrocyte proteinases in the generation of peptides. It is suggested that oxygen and phenylhydrazine free radicals produced in the course of hemoglobin oxidation might be responsible for protein fragmentation. We also discuss a possible relationship between the fragmentation of oxidized hemoglobin and the ATP-independent proteolysis stimulated by oxidative agents.