Kinetic Evidence for a Tetrameric Functional Unit in Hemoglobin

Q H Gibson,L J Parkhurst

doi:10.1016/s0021-9258(17)48178-6

Abstract

Abstract 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.

Highlights

This limiting value was used as a base-line for calculating the quantity shown in Fig. 3 as rapid hemoglobin, which was obtained by expressing the proportion of rapid hemoglobin at any time after initiation of the flow reaction as a percentage of the proportion of rapid hemoglobin observed under base-line conditions
The combination velocity for carbon monoxide in both flow and flash experiments was decreased by a small amount over the values obtained in 0.05 M
It is clear that interactions within a tetrameric unit must be associated with the development of rapid behavior in hemoglobin on partial flash photolysis. Follow that these are the only interactions in hemoglobin, and interactions within dimer units are necessary for a description of the kinetic and equilibrium results already cited [3, 5, 7], the intradimer interactions would be subsidiary rather than primary in explaining ligand binding

Summary

From the Section of Biochemistry and Molecular

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. Guidotti [7] has proposed that ligand molecules combine effectively in pairs: that is, when a ligand molecule binds to one of a pair of sites the affinity of the other site is much increased, and Parkhurst and Gibson [8] have represented kinetic results by a dimer model in which the increase in affinity follows, rather than precedes, combination of a second ligand molecule These two dimer models make very different predictions about the time course of the appearance of rapid behavior on flash photolysis, which Gibson and Antonini [9] tried to test by combining stopped-flow and flash techniques. The apparatus they used, which had been designed for work with cytochrome oxidase, required the use of very dilute solutions of hemoglobin, and the effects observed were consistent with a dissociation phenomenon following ligand binding. The photolysis flash initiated the collection of absorbance data using a crystal-controlled timing unit, a PDP8/S computer, and 138E analogue to digital converter (Digital Equipment Corporation, Maynard, Massachusetts)

Tetrameric Hemoglobin

Findings

Time msec