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Biochem J 2004 Dec 1;384(Pt 2):367-75

Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation.

Jia Y, Ramasamy S, Wood F, Alayash AI, Rifkind JM

Alayash AI, US FDA, Ctr Biol Evaluat & Res, Div Hematol, Lab Biochem & Vasc Biol, Bethesda, MD 20892 USA US FDA, Ctr Biol Evaluat & Res, Div Hematol, Lab Biochem & Vasc Biol, Bethesda, MD 20892 USA NIA, Mol Dynam Sect, NIH, Baltimore, MD 21224 USA


O-R-polyHbA(0) is an intra- and intermolecularly O-raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA(0)), O-R-polyHbA(0) was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA(0)) and no measurable cooperativity (h=1). The kinetics of oxygen and CO binding to the protein indicate lower 'on' rates and faster 'off' rates than HbA(0) and the absence of effects of inositol hexaphosphate (IHP) on the kinetics. Other properties consistent with a T-like conformation are inaccessibility of the betaCys-93 thiol group of O-R-polyHbA(0), the hyperfine splitting from nitrogen in the EPR spectrum of the Fe(II)NO complex of O-R-polyHbA(0) and decreased flexibility in the distal haem pocket, as indicated by low-spin bis-histidine complexes detected by EPR of oxidized chains. A comparison of the properties of O-R-polyHbA(0) with those of HbA(0) with and without IHP, as well as the reaction of nitrite with deoxygenated haemoglobin, provide additional insights into the variations in the conformation of T-state haemoglobin in solution (modifications of the T state produced by adding organic phosphates, like IHP and 2,3-diphosphoglycerate). Although the physiological ramifications of locking HbA(0) in the T conformation with the O-raffinose are still unknown, valuable insights into haemoglobin function are provided by these studies of O-R-polyHbA(0).

Category: Journal Article, Peer
PubMed ID: #15303971
PubMed Central ID: #PMC1134120
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2011-10-04 Entry Last Modified: 2012-08-29