title: Structural and functional characterization of glutaraldehyde-polymerized bovine hemoglobin and its isolated fractions.
abstract: Glutaraldehyde-polymerized bovine hemoglobin (PolyHbBv, trade name Oxyglobin), is a non-site-specific modified hemoglobin-based oxygen-carrying solution, developed for use in veterinary medicine. PolyHbBv was fractionated into four distinct tetrameric and multiple polytetrameric forms ranging in molecular mass (87.2-502.3 kDa) using size exclusion chromatography (SEC) and verified by laser light scattering. We evaluated the structural modification occurring in the fractionated mixture of PolyHbBv and assessed the functionality and redox stability of each fraction in relation to the mixture as a whole. Intramolecular cross-linking evaluation as performed by MALDI-MS and SEC under dissociating conditions revealed no-site-specific tetramer stabilization within the fractions; Intermolecular cross-linking was highly correlated with lysine and histidine modification as determined by amino acid composition analysis. While native unmodified hemoglobin, HbBv, PolyHbBv, and PolyHbBv fractions (F1-F4) revealed significant methionine oxidation, modification, or both, the critical betaMet55 located in the functionally plastic domains (alpha1-beta1 interface) of HbBv was unaltered. Moreover, neither of the two betaCys93 located in the highly plastic alpha1-beta2 interface were modified in PolyHbBv or in F1-F4. Our structural analysis also revealed that the reported loss in sensitivity to chloride in PolyHbBv could not be attributed to direct alteration of chloride ion binding amino acids. Structural modification imparted by glutaraldehyde resulted in nearly identical functional characteristics of PolyHbBv and its fractions with regard to oxygen equilibrium, ligand binding, and autoxidative kinetics.
authors.affiliation: 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.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Buehler
authors.forename: Robert A
authors.initials: RA
authors.lastname: Boykins
authors.forename: Yiping
authors.initials: Y
authors.lastname: Jia
authors.forename: Scott
authors.initials: S
authors.lastname: Norris
authors.forename: Darón I
authors.initials: DI
authors.lastname: Freedberg
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002712:Chlorides / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods; D005976:Glutaral / Q000737:chemistry*; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry*; D007834:Lasers; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010316:Particle Size; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D000596:Amino Acids; D002712:Chlorides; D006454:Hemoglobins; C402601:hemoglobin glutamer-200; D010100:Oxygen; D005976:Glutaral