Effect of A3[6]βGlu → Val mutation on reactivity of the CysF9[93]β sulfhydryl group of human haemoglobin S

Abstract

The pH dependence profiles of the apparent second-order rate constant, k_app, for the reaction of the oxy, carbon monoxy and aquomet derivatives of human haemoglobin S with 5,5′-dithiobis(2-nitrobenzoate)(DTNB) in buffers of ionic strength 50 mmol dm^–3 are complex. The pK_as of the ionizable organic phosphate binding groups which influence the reactivity of the CysF9[93]β sulfhydryl group have been determined from quantitative analyses of the complex profiles. These pK_a values were not significantly different from those of haemoglobin A. In the presence of inositol hexakisphosphate (inositol-P₆) each profile assumes a simple form resembling the titration curve of a diprotic acid. The pK_as of HisHC3[146]β and CysF9[93]β were determined from quantitative analyses of the simple profiles. The mean values obtained, 6.6 ± 0.2 and 8.84 ± 0.04, respectively, were the same as those of haemoglobin A. Comparison of the k_app data for haemoglobin S with those of haemoglobin A shows that, irrespective of the presence or absence of inositol-P₆ presence, the carbon monoxy and aquomet derivatives of haemoglobin A react more rapidly than the corresponding haemoglobin S derivatives. In contrast, in the absence of inositol-P₆, oxyhaemoglobin A reacts faster than oxyhaemoglobin S; in the presence of the organic phosphate both haemoglobins react at about the same rate. At an ionic strength of 200 mmol dm^–3 in the absence of inositol-P₆, the pH dependence profiles of k_app for the oxy, carbon monoxy and aquomet derivatives of haemoglobins A and S are simple. Quantitative analyses of these profiles give mean pK_a values of 5.4 ± 0.1 and 8.9 ± 0.2 for HisHC3[146]β and CysF[93]β, respectively. The oxy and carbon monoxy derivatives of both haemo-globins react at about the same rate, but aquomethaemoglobin A reacts significantly more rapidly than aquomethaemoglobin S.