Production of hematinic acid by the reaction of hemoglobin with phenylhydrazine: evidence for the oxidative cleavage of heme.

Incubation of hemoglobin (Hb, 2 mM as heme) with phenylhydrazine (PH, 5-20 mM) under aerobic conditions produced hematinic acid (HA), one of the oxidatively degraded pyrroles derived from protoporphyrin in Hb. However, incubation with H2O2 resulted in a much lower production of HA. HA was determined by gas-liquid chromatography (GLC) after derivatization to the methyl ester. HA produced by PH was dependent on PH concentration and on incubation time. The maximal yield of HA was 18.8 x 10(-4)% in the reaction with 20 mM of PH for 2 h. Degraded compounds (not identified) other than HA derived from porphyrin were observed on TLC of the mixture after the reaction. Oxidation of ferrous ion and the globin of Hb by PH, which causes alternation of Hb, have been made on the mechanism responsible for the acquisition of the hemolytic activity of PH. Our findings regarding HA production reveal that the porphyrin of Hb is added to the sites oxidized. This is discussed in relation to the hemolysis.