The Binding of Protons and Inositol Hexakisphosphate to Ligated and Unligated Human Des‐Arg^141α‐hemoglobin

Abstract

Des‐Arg‐141α‐hemoglobin has been prepared and its proton binding behaviour has been studied. It appeared that the difference in protons bound by hemoglobin and des‐Arg141α‐hemoglobin cannot be explained by the mere absence of Arg‐141α. The results indicate that upon removal of Arg‐141α, two lysyl or arginyl residues become masked and two histidyl residues titratable. The Bohr effect of des‐Arg141α‐hemoglobin appear to be lower than that of hemoglobin. We present evidence that this phenomenon is for the greater part due to the absence of a pK shift of Val‐1α upon ligation of des‐Arg141α‐hemoglobin. This follows from a study of the pH‐dependence of the rate of reaction of 1‐fluoro‐2,4‐dinitro‐benzene with the amino group of Val‐1α. In the presence of inositol hexakisphosphate the Bohr effect of des‐Arg141α‐hemoglobin is still smaller than that of hemoglobin in the presence of this polyanion. Above pH 7 the proton uptake by des‐Arg141α‐hemoglobin upon binding of inositol hexakisphosphate is smaller than that observed for hemoglobin. The possibility is discussed that the change in proton binding behaviour of hemoglobin upon removal of Arg‐141α is due to a conformational change in the FG region of the α chains. As a result His‐89α becomes titratable and Lys‐90α or Arg‐92α masked. This change in conformation might also be the reason for the high oxygen affinity and the low cooperativity of des‐Arg141α‐hemoglobin.