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

Belur N. Manjula, Ashok Malavallin, Paul K. Smith, Nei Li Chan, Arthur Arnone, Joel M. Friedman, A. Seetharama Acharya

Research output: Contribution to journalArticle

33 Citations (Scopus)

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(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.

Original languageEnglish (US)
Pages (from-to)5527-5534
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number8
DOIs
StatePublished - Feb 25 2000

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Hemoglobin A
Hemoglobins
Salts
2,3-Diphosphoglycerate
Genetic engineering
Recombinant DNA
Chlorides
Acetates
Stabilization
polyethylene glycol 2000
bis(maleidophenyl)PEG2000
Technology
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cys-93-ββ-succinimidophenyl polyethylene glycol 2000 hemoglobin A : Intramolecular cross-bridging of hemoglobin outside the central cavity. / Manjula, Belur N.; Malavallin, Ashok; Smith, Paul K.; Chan, Nei Li; Arnone, Arthur; Friedman, Joel M.; Acharya, A. Seetharama.

In: Journal of Biological Chemistry, Vol. 275, No. 8, 25.02.2000, p. 5527-5534.

Research output: Contribution to journalArticle

Manjula, Belur N. ; Malavallin, Ashok ; Smith, Paul K. ; Chan, Nei Li ; Arnone, Arthur ; Friedman, Joel M. ; Acharya, A. Seetharama. / Cys-93-ββ-succinimidophenyl polyethylene glycol 2000 hemoglobin A : Intramolecular cross-bridging of hemoglobin outside the central cavity. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 8. pp. 5527-5534.
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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(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.",
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AU - Smith, Paul K.

AU - Chan, Nei Li

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AU - Friedman, Joel M.

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