Cys-93-ββ-Succinimidophenyl Polyethylene Glycol 2000 Hemoglobin A

Abstract

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(␤) 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 ϳ60%. 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 deoxyBis-Mal-PEG2000 HbA to be similar to deoxy-HbA but for the loss of the salt bridge between Asp-94(␤) and His-146(␤). 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 crossbridges" 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 Hbs generated by recombinant DNA technology for applications as Hb based therapeutics.Intramolecular cross-bridging of Hb to prevent the dissociation of the tetramers into their constituent ␣␤ dimers has played an important role in the dissection of the molecular aspects of its allostery and cooperativity in oxygen binding (1). Intramolecularly cross-bridged Hbs are also under very active investigation as potential candidates for Hb-based therapeutics (2-4).Several intramolecular cross-bridging approaches have been developed over the years for the stabilization of Hb tetramer against dissociation into ␣␤ dimers (1, 5-10). By and large, the currently available intramolecular cross-bridging reagents are targeted to the reactive functional groups within the central cavity of Hb in domains that interact with allosteric effectors. In fact, some of the cross-linkers have been designed to mimic the interactions of the allosteric effectors. Their design strategy has focused on the complementarity between the stereochemistry of the tagging ends of the proposed bi-or tri-functional reagents and the reactive functional groups within the central cavity (1, 5-10). In view of their location within the central cavity in the modified Hbs, these cross-links offer steric hindrance for interactions with the allosteric effectors compared with unmodified HbA.An alternate plausible approach to introduce intramolecular cross-bridging into Hb is to place the cross-bridges outside the central cavity, thereby avoiding perturbation of the domains of Hb that interact with allosteric effectors. A number of side chain functional groups (sulfhydryl, amino, and carboxyl groups) of the surface residues o...

Dynamics of Allostery in Hemoglobin: Roles of the Penultimate Tyrosine H bonds

“…(Coupling between the bY145F side-chain and the Fe-His bond has been discussed by Friedman and co-workers in the context of modification to the b93Cys residue. 53,54 ) The influence of the aY140F replacement extends further, to the E and A helices, as evidenced by the intensity increase of the 1560 cm K1 UVRR band associated with the internal tryptophan residues, 5 located on the A helices (Figure 1(a)). This intensification signals a strengthening of the A/E H bonds, an effect observed in both deoxyHb and HbCO ( Figure 5(a)).…”

Influence of the chemistry of conjugation of poly(ethylene glycol) to Hb on the oxygen-binding and solution properties of the PEG-Hb conjugate

“…The determination of PEGylation-induced size enhancement of Hb by SEC on Superose 12 columns (Amersham Biosciences), RPHPLC analysis of globin chains on a Vydac C4 column (4.6 × 250 mm), oxygen-affinity measurements and SDS/PAGE were all carried out as previously described [26]. The extent of PEGylation of HbA was measured by determining the freeamino groups remaining on the protein by titration with 2,4,6-TNBS (2,4,6-trinitrobenzenesulphonic) acid [27,28].…”

Molecular Aspects of the High Oxygen Afinity of Non-Hypertensive Hexa Pegylated Hemoglobin, [(SP-PEG5K)6-Hb]

“…Our laboratory has been developing new PEGylation protocols with a view to develop cost-effective PEGylation approaches to design and generate novel PEGylated Hbs [7][8][9][10][11][12]. One of the approaches is PEGylation based on maleimide chemistry.…”