Structural and oxygen-binding properties of divinyl-sulfone-reacted bovine hemoglobin as a function of protein ligation state and reactant concentration.
Bovine hemoglobin (HbBv) was reacted with divinyl sulfone (DVS) at 2-4 degrees C and pH 7.4 for 24 h. Two procedures were employed: (1) low concentration (conc.) of oxygenated HbBv (ligand O2) and low molar ratio of DVS/oxyHbBv (25 ml of 8% oxyHbBv, 0.01 M DVS); (2) low conc. of deoxygenated HbBv (no ligand) and low DVS/deoxyHbBv molar ratio (25 ml of 8% deoxyHbBv, 0.01 M DVS). The nonpolymerized purified products of procedures 1 and 2 were designated oxyHbBv-DVS and HbBv-DVS. Utilizing a high conc. solution of oxyHbBv-DVS and a relatively high molar ratio of DVS/oxyHbBv-DVS (25 ml of 15% oxyHbBv-DVS, 0.03 M DVS) it was possible to aerobically polymerize the modified hemoglobin (procedure 3). Similarly it was possible to anaerobically polymerize HbBv-DVS (25 ml of 15% HbBv-DVS, 0.03 M DVS)(procedure 4). The polymerized products of procedures 3 and 4 were designated oxyPoly HbBv-DVS and Poly HbBv-DVS. The four isolated products were characterized structurally using SDS-PAGE and gel-permeation HPLC and functionally employing a Hemox-analyzer at 37 degrees C, pH 7.4 and 0.15 M Cl. Both oxyPoly HbBv-DVS (P50 = 13 mm Hg, n = 1.1) and Poly HbBv-DVS (P50 = 61 mm Hg, n = 1.6) were shown to be mixtures of intermolecularly-crosslinked hemoglobin. OxyHbBv-DVS (P50 = 13 mm Hg, n = 1.3) was shown to be an intramolecularly-crosslinked 64 kDa material, whereas HbBv-DVS (P50 = 52 mm Hg, n = 1.9) was found to be an intramolecularly-modified 64 kDa derivative, but not an intramolecularly-crosslinked one.(ABSTRACT TRUNCATED AT 250 WORDS)
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Artificial Cells and Organs Research Centre Faculty of Medicine, McGill University, Montréal, Québec, Canada.
D000818:Animals; D001802:Blood Substitutes / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism*; D013450:Sulfones / Q000737:chemistry / Q000378:metabolism*
D001802:Blood Substitutes; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D008024:Ligands; D013450:Sulfones; C081606:bovine hemoglobin-divinyl sulfone; C081607:polymerized bovine hemoglobin-divinyl sulfone; D010100:Oxygen