Kinetic Evidence for a Tetrameric Functional Unit in Hemoglobin

The time course of the appearance of rapid hemoglobin, as judged by the results of partial photodissociation of carbon monoxide, was followed with a stopped-flow flash photolysis apparatus. Deoxyhemoglobin was mixed with carbon monoxide and a photolysis flash was fired after a short accurately measured time interval. There is a marked lag in the appearance of rapid hemoglobin. Analysis of the results suggests that this is not due to a first order process following ligand binding. The lag cannot, therefore, be due to dissociation of hemoglobin into subunits or to loss of 2,3-diphosphoglycerate. The results appear to be incompatible with schemes having the dimer as the prime unit of function. It is proposed that the change in reactivity associated with the appearance of rapid hemoglobin occurs after at least three ligand molecules have combined. This conclusion is supported by experiments with the hybrid species αNOβCO which behaves as a functional tetramer. The time course of the appearance of rapid hemoglobin was not significantly influenced by 2,3-diphosphoglycerate.