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 proce-dure developed by Benesch and Benesch (1). The reaction of bovine Hb with NFPLP leads to a cross-linkag between the subunits, which greatly stabilizes the lowaffinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into abdimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confrontedwith two different structural alternatives; the cross-link of b chains may be formed between lysine 82 of b2 andthe N terminus of methionine 2 of b1or between the two lysine 82 residues of both b2chains. Digestion of modified bglobin chains and isolation of the cross-linkedpeptide have showed that NFPLP cross-links Met-b2 and Lys-b82. 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 sustitute.