Cys-93-ββ-succinimidophenyl polyethylene glycol 2000 hemoglobin A -: Intramolecular cross-bridging of hemoglobin outside the central cavity

Bis(maleidophenyl)-PEG2000 (Bis-Mal-PEG2000), a new bifunctional protein cross-linker targeted to sulfhydryl groups, introduces intra-tetrameric cross-links into oxy-HbA in nearly quantitative yields. Structural as well as crystallographic analyses of the cross-linked species, Bis-Mal-PEG2000 HbA, identified Cys-93(beta) as the site of intramolecular cross-linking. The cross-bridging had only a limited influence on the O-2 affinity and cooperativity of HbA in 50 mM BisTris acetate, pH 7.4. However, the Bohr effect was reduced by similar to 60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effecters 2,3-diphosphoglycerate, IHP, and chloride, albeit to a lesser degree compared with HbA, Crystallographic analysis revealed the overall structure of deoxy-Bis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(beta) and His-146(beta), The large influence of the cross-bridging on the alkaline Bohr effect of HbA is consistent with the loss of this salt bridge. Unlike the central cavity cross-bridges described previously, the cross-link introduced by Bis-Mal-PEG2000 into HbA is an outside the central cavity cross-bridge. In view of its oxy-conformational specificity and limited influence on O-2 affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O-2 affinity Rbs generated by recombinant DNA technology for applications as Hb based therapeutics.