Binding of beta-Naphthyl Triphosphate to Human Adult Hemoglobin Accompanying Deoxygenation. Investigated by Simultaneous Measurements of Fluorescence, Absorbance and Partial Pressure of Oxygen

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Abstract
Binding of a fluorescent allosteric effector, .beta.-naphthyl triphosphate (.beta.-NapP3), to human adult Hb (HbA) at various levels of O2 saturation were investigated by simultaneous measurements of fluorescence, absorbance and O2 partial pressure. Amounts of .beta.-NapP3 bound to HbA were easily estimated from the fluorescence intensities of HbA solutions, because the fluorescence of .beta.-NapP3 bound to HbA is completely quenched. Exchange reactions of the above fluorescent allosteric effector with 2,3-diphosphoglycerate (DPG) were also examined at various levels of O2 saturation. .beta.-NapP3 binds to deoxyHbA tetramer in the molar ratio of 2:1, and 1 of the 2 .beta.-NapP3 competes with DPG. .beta.-NapP3 binds to completely oxygenated HbA tetramer in the molar ratio of 1:1, and the bound .beta.-NapP3 was not released by adding DPG. The binding affinity of .beta.-NapP3 for the noncompetitive site of completely oxygenated HbA, to which DPG does not bind, was smaller than that for the noncompetitive site of deoxyHbA, to which DPG also does not bind. The correlations between O2 bindings by HbA and the bindings of .beta.-NapP3 to HbA in the intermediate stages of deoxygenation were investigated. HbA as a tetramer exists in 3 conformational states rather than simple 2 states as Monod, Wyman and Changeux had proposed.