The γ-carboxyl groups of Glu-43(β) and Glu-22(β) of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide at p H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence of N-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22(β) and Glu-43(β) with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43(β) with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43(β) (i.e., chemical reactivity) as a function of the pH in the region of 5.5-7.5, the apparent pKa of its γ-carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43(β) of HbS at pH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(β), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the γ-carboxyl group and could be one of the crucial preludes to the polymerization process.
- Hemoglobin S
- ionization behavior
- rescuing of O-acyl isourea adduct
- salt bridge
ASJC Scopus subject areas