Kinetics of the Reaction of Hemoglobin with Ethylisocyanide

The kinetics of the reaction of human deoxyhemoglobin with ethylisocyanide has been studied, by rapid mixing, over a 50- to 100-fold range of ligand concentration, both as a function of protein concentration (from 3 to 30 x 10-6 m) and ionic strength (from 0.2 to 2.2 m). The results show that the progress curve, which is autocatalytic at high ligand concentration, tends to change shape as the ethylisocyanide concentration is decreased, and finally becomes markedly diphasic. The experimental results can be fitted satisfactorily with a simple dimer scheme, with only two combination and two dissociation velocity constants. Consideration of these results, in conjunction with other data, allows us to arrive at important conclusions concerning the kinetic origin of co-operativity as observed at equilibrium. The most significant of these is that, to a major degree, cooperative ligand binding finds its kinetic justification in a large decrease of the dissociation velocity constant as the reaction proceeds.