Effects of cross-linking and zero-link polymerization on oxygen transport and redox chemistry of bovine hemoglobin
Laboratory of Biochemistry and Vascular Biology (LBVB), Division of Hematology, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), MD 20892, USA.Biochimica et Biophysica Acta (Impact Factor: 4.66). 06/2009; 1794(8):1234-42. DOI: 10.1016/j.bbapap.2009.04.008
Cross-linked hemoglobins (Hbs) were found to have enhanced oxidative reactions which compromise the ability of cell-free Hb to carry oxygen. Zero-link bovine Hb (ZL-HbBv), also known as OxyVita, a large polymer held together by pseudopeptide bonds on the surface of adjacent tetramers, provides a model in which these reactions can be evaluated. The oxygen affinity of ZL-HbBv was greatly increased, whereas the oxygen binding cooperativity (n(50)) as well as the regulatory responses to pH and chloride ions was diminished. Rapid mixing kinetic studies revealed faster carbon monoxide (CO) and nitric oxide (NO) binding to ZL-HbBv, consistent with a more accessible heme pocket conformation. The rate of autoxidation of ferrous ZL-HbBv was 3 folds faster than the unmodified HbBv (control) but only slightly suppressed by the presence of superoxide dismutase and catalase enzymes. The peroxide (H(2)O(2)) reaction rates of ferric ZL-HbBv and its degradation were comparable to that of the control. The rate of heme loss from ZL-HbBv to a mutant apomyoglobin (H64Y/V68F) was also very close to that of the control. Taken together, allosteric and redox reactions of this protein are altered due to heme accessibility to solvent, however, the compact tetramer to tetramer interaction of the ZL-HbBv polymer appears to restrict heme loss even in the presence of excess H(2)O(2).
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ABSTRACT: The development of oxygen (O2)-carrying blood substitutes has evolved from the goal of replicating blood O2 transport properties to that of preserving microvascular and organ function, reducing the inherent or potential toxicity of the material used to carry O2, and treating pathologies initiated by anemia and hypoxia. Furthermore, the emphasis has shifted from blood replacement fluid to "O2 therapeutics" that restore tissue oxygenation to specific tissues regions. This review covers the different alternatives, potential and limitations of hemoglobin-based O2 carriers (HBOCs) and perfluorocarbon-based O2 carriers (PFCOCs), with emphasis on the physiologic conditions disturbed in the situation that they will be used. It describes how concepts learned from plasma expanders without O2-carrying capacity can be applied to maintain O2 delivery and summarizes the microvascular responses due to HBOCs and PFCOCs. This review also presents alternative applications of HBOCs and PFCOCs namely: 1) How HBOC O2 affinity can be engineered to target O2 delivery to hypoxic tissues; and 2) How the high gas solubility of PFCOCs provides new opportunities for carrying, dissolving, and delivering gases with biological activity. It is concluded that the development of current blood substitutes has amplified their applications horizon by devising therapeutic functions for O2 carriers requiring limited O2 delivery capacity restoration. Conversely, full, blood-like O2-carrying capacity reestablishment awaits the control of O2 carrier toxicity.
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ABSTRACT: Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS-PAGE, SEC, and ESI-MS), susceptibility to oxidation (k(ox)), responses to physiological conditions (pH, [Cl(-)], [IHP], and T), and ligand binding kinetics (O(2), NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF-purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb-based O(2) carrier synthesis.
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