Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation.
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).
Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, Maryland 20892, USA.
Yiping
Y
Jia
Somasundaram
S
Ramasamy
Francine
F
Wood
Abdu I
AI
Alayash
Joseph M
JM
Rifkind
D000494:Allosteric Regulation / Q000502:physiology; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006442:Glycated Hemoglobin / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008706:Methemoglobin / Q000737:chemistry; D008968:Molecular Conformation; D009569:Nitric Oxide / Q000737:chemistry; D009573:Nitrites / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000737:chemistry; D011887:Raffinose / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D003432:Cross-Linking Reagents; D006442:Glycated Hemoglobin; D006454:Hemoglobins; D008706:Methemoglobin; D009569:Nitric Oxide; D009573:Nitrites; D010100:Oxygen; D010108:Oxyhemoglobins; D011887:Raffinose; D013438:Sulfhydryl Compounds; C058377:hemoglobin A, glycosylated-nitric oxide complex