ORGANIC PHOSPHATE-BINDING GROUPS ELECTROSTATICALLY LINKED TO THE REACTIVITY OF THE CYS F9 [93]BETA SULFHYDRYL-GROUP OF HEMOGLOBIN

At an ionic strength of 0.05 mol dm(-3) the pH-dependence profile for the reaction of the Cys F9 [93] beta sulfhydryl group of human haemoglobin (stripped of organic phosphates) with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) is complex. In the presence of a four-fold molar excess of inositol hexakisphosphate (inositol-P-6) over haemoglobin tetramers, the pH-dependence profile changes dramatically from a complex to a simple form resembling the titration curve of a diprotic acid. Values of the apparent second-order rate constant, k(app) are also drastically reduced. Quantitative analyses of the simple profiles indicate that the reactivity of the sulfhydryl is linked to the ionization of two amino acid residues on the protein, with pK(a) values around 6.6 and 8.7. These pK(a) s are assigned to His HC3 [146] beta and Cys F9 [93] beta, respectively. Since inositol-P-6 simplifies the complex pH-dependence profile obtained for stripped haemoglobin by binding to the cationic groups at the organic phosphate binding site, we have analysed the complex pH-dependence profile of stripped haemoglobin quantitatively by assuming that there is an electrostatic interaction between the sulfhydryl and the cationic groups. From the analyses of the complex profiles for the oxy, carbon monoxy, azidomet and cyanomet derivatives, mean pK(a) values of 6.4 +/- 0.1, 7.5 +/- 0.2 and 9.5 +/- 0.02 are obtained for the groups that are electrostatically linked to the Cys F9 [93] beta sulfhydryl group. The pK(a) of 6.4 is assigned to His NA2 [2] beta and HisH21 [143] beta; the pK(a) of 7.5 is assigned to Val NAI [1] beta; and the pK(a) of 9.5 is assigned to Cys F9 [93] B. For aquomethaemoglobin, analysis shows that, in addition to these groups, the water molecule attached to the sixth coordination position of the iron(III) atom is also electrostatically linked to the sulfhydryl.