Abstract
We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2′-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2′-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-α-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O.−2 and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Kb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.
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