Organic phosphate-binding groups electrostatically linked to the reactivity of the Cys F9 [93]β sulfhydryl group of haemoglobin

Abstract

At an ionic strength of 0.05 mol dm^–3 the pH-dependence profile for the reaction of the Cys F9 [93]β 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-P₆) 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_as are assigned to His HC3 [146]β and Cys F9 [93]β, respectively.

Since inositol-P₆ 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]β sulfhydryl group. The pK_a of 6.4 is assigned to His NA2 [2]β and HisH21 [143]β; the pK_a of 7.5 is assigned to Val NA1 [1]β; and the pK_a of 9.5 is assigned to Cys F9 [93]β. 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.