Chain Inequivalence in Bovine Methemoglobin

Abstract

Using pulse radiolysis, a single heme in the tetramer of bovine methemoglobin was reduced within a few microseconds to the ferro state, producing a valence intermediate. The kinetics of oxygen binding to the valence intermediate as well as the re-oxidation of the ferro-heme to the ferric state were studied as a function of pH.The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectively). A sigmoidal curve representing a transition between the two states as a function of pH was derived.Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant.The reaction between the valence intermediate and ferricyanide at pH 7.75 (R state) consisted of two (about) equal contributions (kl = 23 x lo4 M-' s-'; k2 = 2.1 x lo4 M-' s-' ) attributed to the p and a subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was [4] also revealed that p chains are more reactive towards various ligands than a chains. In this investigation we have studied the oxidation of partly reduced bovine methemoglobin by ferricyanide and revealed chain inequivalence.Oxidation of human hemoglobin by ferricyanide has been studied by Antonini et al.[lo] using a stoppedflow apparatus. They found that below pH 8 the rate of oxidation is reduced as the reaction proceeds and they attributed this phenomenon to a possible intramolecular heterogeneity between a and p chains.The oxidation of tetrameric hemoglobin with ferricyanide is widely used for the preparation of methemoglobin. The reaction proceeds by a single electron transfer from the heme iron to ferricyanide producing oxidized ferric heme. Although the overall reaction has been studied in great detail [6] less information is available concerning the oxidation of individual chains within the tetramer. It is evident that non-equivalence of the subunits will be observed most clearly when the hemoglobin molecules are either fully in the high-affinity state or all in the lowaffinity state.The methemoglobin molecule is visualized as residing in either of two sets of quaternary structures, in each set the structures are closely related. In recent studies [ l l , 121 we have suggested that by using the pulse radiolysis technique the quaternary structure of methemoglobin can be characterized by following the kinetics of CO and O2 binding to its valence intermediate. The rate constants for these reactions were found to be dependent upon the pH and the presence of organic phosphates [ll, 121. The changes in these rate constants indicated a transition between affinity states representing a switch between quaternary structures.In this study the affinity state of bovine methemoglobin is monitored by studying the oxygenation of its valence intermediate. The changes of the oxygenation process enable us to characterize the conditions at which the methemoglobin molecule resides in either