PROBING THE HEMOGLOBIN CENTRAL CAVITY BY DIRECT QUANTIFICATION OF EFFECTOR-BINDING USING FLUORESCENCE LIFETIME METHODS

Time-resolved fluorescence methods have been used to show that 8-hydro xy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosp hoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO ) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and similar to 27 ps, respectively, was developed to measure the binding affinity of thi s probe. HPT binds to a single site and is displaced by inositol hexap hosphate at a 1:1 mol ratio, indicating that binding occurs at the 2,3 -diphosphoglycerate site in the central cavity. Furthermore, the resul ts imply that low pH HbACO exists as an altered R state and not an equ ilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the bindi ng site in several cross-bridged HbA derivatives.