Effects of polymerization on the oxygen carrying and redox properties of diaspirin cross-linked hemoglobin

Abstract

Human hemoglobin site specifically cross-linked with bis(3,5-dibromosalicyl)fumarate results in a low oxygen affinity hemoglobin-based red cell substitute (α-DBBF). Polymerization of α-DBBF by bis(maleoylglycylamide) polyethylene glycol (BMAA-PEG) yields poly α-DBBF which offers the added benefits of reduced renal clearance and increased retention in the vascular circulation. Oxygen equilibrium curves for poly α-DBBF are slightly left-shifted (higher O 2 affinity) compared to those of α-DBBF; with a diminished cooperativity and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide binding), poly α-DBBF exhibits a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. The rate of nitric oxide binding to the oxidized form of poly α-DBBF shows little or no change compared to the intramolecularly cross-linked derivative. The reduction of cyanomet poly α-DBBF by dithionite is several fold faster than that of HbA 0 and α-DBBF whereas the slow subsequent cyanide dissociation from the ferrous iron remained unchanged among all proteins. The propensity of poly α-DBBF for auto-oxidation is slightly enhanced over α-DBBF whereas the extent of oxidative modification by hydrogen peroxide is very similar. Polymerization appears to selectively modify ligand interactions and redox kinetics of the tetrameric cross-linked form which reflects a possibly more open heme pocket. The data suggests that changes in oxygenation properties of hemoglobin brought about by a given modification are not necessarily predictive of other functional changes.