| Autor: |
Manjula, B N, Malavalli, A, Smith, P K, Chan, N L, Arnone, A, Friedman, J M, Acharya, A S |
| Zdroj: |
Journal of Biological Chemistry; February 2000, Vol. 275 Issue: 8 p5527-34, 8p |
| Abstrakt: |
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 approximately 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(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 Hbs generated by recombinant DNA technology for applications as Hb based therapeutics. |
| Databáze: |
Supplemental Index |
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