Use of a Fluorescent Analogue of 2,3-Diphosphoglycerate as a Probe of Human Hemoglobin Conformation during Carbon Monoxide Binding

Abstract

Abstract The interaction of human hemoglobin (Hb) with 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT) was studied at pH 6.0 and T/2 = 0.04. Binding of HPT was determined by quenching of its fluorescence upon interaction with Hb. HPT binds to deoxy-Hb with a stoichiometry of 1 mole per mole of Hb tetramer and with a higher affinity for the deoxy than the liganded form analogous to the binding of the physiologically important 2,3-diphosphoglycerate (DPG). Binding is prevented by either DPG or inositol hexaphosphate. A qualitative comparison of the time courses of HPT release and CO combination during the reaction of CO with deoxy-Hb indicates a marked lag in the release of HPT compared with CO binding. This led to the conclusion that during the course of CO binding, significant amounts of partially liganded Hb intermediates are formed and the release of HPT from the intermediates does not occur in proportion to the amount of CO bound. Quantitative analysis of the data, in the form of least squares fitting procedures, was carried out to examine the ability of some elementary models to describe the results. The best fit was obtained on the assumption that HPT is released after 3 CO molecules have bound to hemoglobin.