Bovine hemoglobin cross-linked through the beta chains - Functional and structural aspects

2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch (1). The reaction of bovine Db with NFPLP leads to a cross-linkage between the beta subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into alpha beta dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O-2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross-link of beta chains may be formed between lysine 82 of beta(2) and the N terminus of methionine 2 of beta(1) or between the two lysine 82 residues of both beta(2) chains. Digestion of modified beta globin chains and isolation of the cross-linked peptide have showed that NFPLP cross-links Met-beta 2 and Lys-beta 82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute.