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(β) as the site of intramolecular cross-linking. The cross- bridging had only a limited influence on the O₂ affinity and cooperativity of HbA in 50 mM BisTris acetate, pH 7.4. However, the Bohr effect was reduced by ~60%. Bis-Mal-PEG2000 HbA retained sensitivity to the presence of allosteric effectors 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(β) and His-146(β). 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 O₂ affinity, this new cross-linking strategy holds promise for the stabilization of new designer low O₂ affinity Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.