Effect of A3[6]beta(Glu->Val) mutation on reactivity of the CysF9[93]beta sulfhydryl group of human haemoglobin S

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(a) s of the ionizable organic phosphate binding groups which influence the reactivity of the CysF9[93]beta 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-P-6) each profile assumes a simple form resembling the titration curve of a diprotic acid. The pK(a) s of HisHC3[146]beta and CysF9[93]beta 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-P-6 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-P-6, 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-P-6, 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]beta and CysF9[93]beta, respectively. The oxy and carbon monoxy derivatives of both haemoglobins react at about the same rate, but aquomethaemoglobin A reacts significantly more rapidly than aquomethaemoglobin S.