The gamma-carboxyl groups of Glu-43(beta) and Glu-22(beta) of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide at pH 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(beta) and Glu-43(beta) 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(beta) 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(beta) (i.e., chemical reactivity) as a function of the pH in the region of 5.5-7.5, the apparent pKa of its gamma-carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43(beta) of HbS at pH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(beta), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the gamma-carboxyl group and could be one of the crucial preludes to the polymerization process.