Cys-93-betabeta-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 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.
Manjula
B N
BN
Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. manjula@aecom.yu.edu
Malavalli
A
A
Smith
P K
PK
Chan
N L
NL
Arnone
A
A
Friedman
J M
JM
Acharya
A S
AS
D002021:Buffers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008301:Maleimides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors
D002021:Buffers; D003432:Cross-Linking Reagents; D008301:Maleimides; D013438:Sulfhydryl Compounds; C405685:bis(maleidophenyl)PEG2000; D011092:Polyethylene Glycols; D006441:Hemoglobin A; D003545:Cysteine; D010100:Oxygen