Structural and Oxygen-Binding Properties of Divinyl-Sulfone-Reacted Bovine Hemoglobin as a Function of Protein Ligation State and Reactant Concentration

Abstract

Bovine hemoglobin (HbBv) was reacted with divinyl sulfone (DVS) at 2–4°C and pH 7.4 for 24 h. Two procedures were employed: (1) low concentration (cone.) 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 cone, 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°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 hemoglobins. 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. The results indicate that intramolecular crosslinkage is achieved only in the presence of oxygen, while high P₅₀ and n values are obtained when the reaction is performed in the absence of the oxygen ligand.