ISOLATION AND STABILITY OF PARTIALLY OXIDIZED INTERMEDIATES OF CARP HEMOGLOBIN - KINETICS OF CO BINDING TO THE MONO-FERRIC AND TRIFERRIC SPECIES

The monoliganded and triliganded forms of the asymmetric valency hybri ds of carp hemoglobin were isolated using high-performance liquid chro matography. These partially oxidized hybrids were shown to be sufficie ntly stable to permit the measurement of the kinetics of CO binding. T he effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO bin ding to the fully ferrous molecule. To a first approximation, the kine tic behavior of the monoferric derivative was consistent with a small shift in the T <-> R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conf ormational equilibrium in favor of the R state. The kinetic behavior o f the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presenc e of cyanide on the ferric hemes than by water. Not all of the data co uld be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivat ive at low pH in the presence of inositol hexaphosphate.