The generation of O2-by the interaction of the hemolytic agent, phenylhydrazine, with human hemoglobin.

Abstract

Phenylhydrazinereacts with adult oxy-or methemoglobin in the presence of molecular oxygen to generate Ot-.The mechanism of the reaction apparently differs in each case.The generation of 0~~ is monitored by following the reduction of ferricytochrome c or the oxidation of epinephrine.The relationship between 011 and hemoglobin is a topic of current interest.The generation of 0, occurs during the autoxidation of shark hemoglobin at pH 6.8 (1) and of bovine hemoglobin in the presence of 1 M phosphate at pH 6.0 (2).Many chemical compounds cause the oxidation of hemoglobin ( 3), yet the underlying molecular mechanisms remain unknown.In certain cases the chemically induced oxidation of hemoglobin might involve the concomitant production of 02.Phenylhydrazine causes the oxidation of hemoglobin, catalyzes the destruction of hemoglobin, and induces hemolysis of red blood cells (4).Phenylhydrazine also generates hydrogen peroxide, an end product of the dismutation of 0, (5), in the presence of hemoglobin (6).We observed the generation of a significant amount of 02 in the interaction of phenylhydrazine and hemoglobin.Since 0, may contribute to peroxidative hemolysis (7), these results suggest that a link exists between the production of 01 in the reaction of phenylhydrazine and hemoglobin and phenylhydrazine-induced hemolysis.Two systems were employed in the investigation of 02 generation in the reaction of phenylhydrazine and hemoglobin.The first involves the reduction of ferricytochrome c to ferrocytochrome c by 0, and inhibition of this reduction by superoxide dismutase (8).The second involves the oxidation of epinephrine to adrenochrome by 01 and inhibition of this oxidation by superoxide dismutase (9).Oxyhemoglobin samples were prepared from hemolysates of adult human blood (10).Methemoglobin was obtained by a procedure using potassium ferricyanide (11).Measurements of the reduction of ferricytochrome c (Biochemical Laboratories CO.,