Effect of ionizing radiation on haemoglobin: the oxy-derivative of haemoglobin Iwate.

It is well established that exposure of oxyhaemoglobin to ionizing radiation results in remarkably selective electron addition to the (FeO2) unit, giving a novel species, (FeO2)-, in which the extra electron is largely localized on iron and dioxygen. This work has now been extended to haemoglobin (Hb.) Iwate. The haemoglobin M. Iwate used is a mutant haemoglobin having only Fe(III) alpha-chains by oxy beta-chains (alpha 2 Met beta 2 oxy). The haem iron atoms in the alpha-chains are coordinated in the fifth site by a proximal tyrosine in place of histidine. This unit is a high-spin Fe(III) with axial symmetry and prominent electron spin resonance (ESR) features in the g = 6 and g = 2 regions. On exposure to 60Co gamma-rays at 77 K, efficient electron addition occurred at both types of iron centre, giving Fe(II) and (FeO2)- units. The former was monitored by the decrease of the g = 6 feature for Fe(III) and the latter by the growth of g-features at 2.254 (gx), 2.149 (gy) and 1.967 (gz). These values are close to those for the FeO2- centre formed in the beta-chains of normal oxyhaemoglobin. On annealing above 77 K, two changes occurred: first there was a small but clear increase in gx and gy, followed by a marked reduction in gx and gy giving g-values close to those for the centre formed directly in the alpha-chains of the normal protein. Finally, this intermediate species gave a centre having gx = 2.310, gy = 2.180 and gz = 1.935. These values are typical of low-spin Fe(III) haemoglobin and are assigned to the protonated complex, Fe(III)O2H. Ultimately at ca. room temperature, this was converted into the high-spin, met-form, with a gain in the g = 6 feature. These results established that the beta-chain centre in Hb. Iwate behave in the same way as isolated beta-chains. They also confirm that electron addition to the oxy-units is facile, even in the presence of Fe(III) units in each tetramer. The results also confirm that electron capture to give (FeO2)- units is not followed by internal electron-transfer to give Fe(II) from the Fe(III) centres in the alpha-chains.

Functional and physicochemical studies of hemoglobin St. Louis beta 28 (B10) Leu replaced by Gln: a variant with ferric beta heme iron.

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.