| Abstract: | The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas. |
