ANALYSIS OF INTERMEDIATE HEMOGLOBINS IN SOLUTIONS OF HEMOGLOBIN PARTIALLY OXIDIZED WITH FERRICYANIDE

The intermediate hemoglobins which were produced by the partial oxidation of hemoglobin with ferricyanide were clearly separated by preparative isoelectric focusing. Two intermediate hemoglobins (IBI and IBII) were identified as α2+β3+ and α3+β2+ valency hybrids by the studies of absorption spectra and inositol hexaphosphate-induced difference spectra. The distribution of oxyhemoglobin, intermediate hemoglobin and methemoglobin in the solutions of hemoglobin (0.05 M bis-Tris with 0.1 M NaCl) which were partially oxidized by ferricyanide was analyzed in the presence or absence of inositol hexaphosphate at pH 6.6-7.8. The proportion of the α2+β3+ valency hybrid was always higher than that of α3+β2+. Inositol hexaphosphate increased this difference, i.e. the level of α2+β3+ rose and that of α3+β2+ fell in the presence of the organic phosphate. At pH regions studied, the proportion of α2+β3+ and α3+β2+ was not significantly altered by a change in pH whether inositol hexaphosphate was present or not. Furthermore, the effects of ionic strength on the proportion of the hemoglobin derivatives were studied in phosphate buffer at pH 7.0. At low ionic strength (10 mM phosphate buffer), the proportion of α3+β2+ was higher than that of α2+β3+. At higher ionic strength (10 mM phosphate buffer with 0.1 M NaCl or 0.1 M phosphate buffer), the proportion of these two intermediate hemoglobins was nearly equal or the proportion of α2+β3+ was higher than that of α3+β2+. The heterogeneity of the proportion of the intermediate hemoglobins was explained by the functional differences of α and β chains in tetrameric hemoglobin in the reaction with ferricyanide, which were influenced by the presence of high concentration of inorganic phosphate, chloride and low concentration of inositol hexaphosphate. The effects of inositol hexaphosphate on the oxidation of hemoglobin by ferricyanide was considered to be due to the changes in hemoglobin conformation notably involving the β chains. © 1979.