Fragmentation of human hemoglobin by oxidative stress produced by phenylhydrazine

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.