High ionic strength or presence of inositol hexakisphosphate reverses the unusually low pKA of CYSH3(125)Β of guinea pig haemoglobin

Abstract

ABSTRACT. The kinetics of the reaction of 5,5'-dithiobis(2-nitrobenzoate) with guinea pig oxy-and carbonmonoxyhaemoglobin are biphasic. The two phases differ in rate by two orders of magnitude. For the fast phase, quantitative analysis of the pH dependence of the apparent second order rate constant, kapp, shows that it has all the characteristics associated with the reaction of CysF9 (93)β, a sulphydryl group that is invariant in all mammalian haemoglobins. The slow kinetic phase is assigned to CysH3(125)β. Quantitative analysis of the pH dependence of kapp for this phase at 50 mM ionic strength gave an unusually low pKa of 6.0 for this sulphydryl group. Published data on guinea pig haemoglobin show that it has a much-enhanced acid Bohr effect compared to human haemoglobin. This indicates that CysH3(125)β functions as an acid Bohr group in guinea pig haemoglobin. Comparison with human haemoglobin indicates that this cysteine reduces the alkaline Bohr effect of guinea pig haemoglobin between pH 7 and 6, and enhances its acid Bohr effect below pH 6. From kinetic data collected at high salt concentration, and in the presence of inositol hexakisphosphate, it is shown that the pKa of the sulphydryl increases to ca 9 and 7.7, respectively. Under these conditions, CysH3(125)β ceases to be an acid Bohr group.