Kinetic studies of oxygenation of a- and b-subunits within pyridoxal 50-phosphate derivatives of human hemoglobin

Different effects on individual properties of a- and b-subunits within oxygenated human hemoglobin (HbA) are revealed at the pyridoxal 50-phosphate modification. The rate constant of bimolecular oxygenation of the a-subunits within triliganded HbA and the apparent quantum yield of photodissociation of the a-subunits within completely oxygenated HbA decrease from 18.8G1.3 (mM s)K1 to 13.2G1.6 (mM s)K1 and from 0.0114G0.0012 to 0.0087G0.0009, respectively. The association rate constant of the b-subunits does not vary distinctly. However, the apparent quantum yield of photodissociation of the b-subunits is found to increase from 0.041G0.004 to 0.067G0.006. A previously proposed approach [B.M. Dzhagarov, S.V. Lepeshkevich, Chem. Phys. Lett. 390 (2004) 59.] is used for determination of the extent of subunit dissociation rate constant difference and the extent of subunit affinity difference from single flash photolysis experiment. The dissociation rate constant for the a-subunits within the tetramer is lowered by a factor of 1.3 with pyridoxylation. Since the association rate constant for the a-subunits are also lowered by a factor of 1.4, the a-subunits affinity to oxygen remains practically unaltered. The dissociation rate constant for the b-subunits is increased by a factor ofw1.6. Because the association rate constant for the b-subunits does not vary virtually, the effect of the pyridoxylation on the oxygenation of the b-subunits is a 1.7-fold decrease in the O2 affinity. Therefore the net pyridoxylation effect on the oxygenation of the liganded HbA is a reduction in the O2 affinity mainly due to the decrease in the b-subunits affinity for oxygen. The present data of the bimolecular oxygenation study are analyzed in conjunction with the data of a previous geminate oxygenation research [S.V. Lepeshkevich, J. Karpiuk, I.V. Sazanovich, B.M. Dzhagarov, Biochemistry 43 (2004) 1675.]. For explanation of the pyridoxal 50- phosphate regulation mechanism of the HbA subunits oxygenation, a parallel pathway model of ligand movement in the protein is favoured. q 2004 Elsevier B.V. All rights reserved.