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

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

“…To test the correctness of this assignment, kinetic experiments were carried out at 200 mM ionic strength. It was previously reported that an increase in ionic strength from 50 to 200 mM practically had no effect on the pK a s of HisHC3(146)β and CysF9(93)β of stripped human haemoglobin [9]. Profiles similar to those reported in Figure 1 were earlier accounted for in terms of the fractional population of the thiol anion form of the sulphydryl [9-11, 19, 21, 22].…”

“…In particular, the author failed to observe the biphasic nature of the pH dependence profile ( Figure 1; Table 1) and, consequently, erroneously assigned the fast kinetic phase to CysH3(125)β instead of CysF9 (93)β. The very high rates obtained for the reaction of DTNB with CysF9(93)β of guinea pig haemoglobin are about 2 orders of magnitude faster than what was previously reported for other haemoglobins [9][10][11]22]. At present there is no explanation for these very high rates.…”

“…The endogenous organic phosphate in red blood cells is 2,3-bisphosphoglycerate (2,3-BPG) but inositol hexakisphosphate (inositol-P 6 ) is used more often in haemoglobin studies. This organic phosphate reduces the reactivity of CysF9(93)β and increases the pK a of HisHC3(146)β in human haemoglobin by 1 pK a unit [9]. Figure 3 shows the pH dependence profiles of k app for the fast kinetic phase of the reaction of DTNB with guinea pig haemoglobin in the presence of inositol-P 6 .…”

“…Only a single pK a of 7.8 was obtained. Since inositol-P 6 increases the pK a of HisHC3(146)β of human haemoglobin by 1 pK a unit [9], the single pK a obtained from the data in Figure 3 suggests that the pK a of this histidine in guinea pig haemoglobin has been increased from 6.5 in stripped haemoglobin (Figure 1; Table 1 of reference 15) to 7.8 in the presence of inositol-P 6 , an increase of 1.3 pK a units. In other words, in the presence of inositol-P 6 the pK a s of HisHC3(146)β and CysF9(93)β of guinea pig haemoglobin coincide.…”

“…Furthermore, Ho and co-workers have concluded that mutations or chemical modifications at the F9(93)β site affect the salt bridge and the organic phosphatebinding site and also destabilize the T quaternary state [8]. By studying the reactivity of CysF9(93)β as a function of pH it is demonstrated that inositol hexakisphosphate (inositol-P 6 ), an organic phosphate, raises the pK a of HisHC3(146)β of human haemoglobins A and S by one pK a unit, thereby strengthening the HisHC3/AspFG1 salt bridge and reducing the reactivity of the sulphydryl group [9]. These interesting findings raise the possibility that sulphydryl groups located at other positions in the haemoglobin molecule might provide equally interesting information.…”