Binding of beta-naphthyl triphosphate to human adult hemoglobin accompanying deoxygenation. Investigated by simultaneous measurements of fluorescence, absorbance and partial pressure of oxygen.
Binding of a fluorescent allosteric effector, beta-naphthyl triphosphate (beta-NapP3), to human adult hemoglobin (HbA) at various levels of oxygen saturation were investigated by simultaneous measurements of fluorescence, absorbance and oxygen partial pressure. Amounts of beta-NapP3 bound to HbA were easily estimated from the fluorescence intensities of HbA solutions, because it was previously proved that the fluorescence of beta-NapP3 bound to HbA is completely quenched. Exchange reactions of the above fluorescent allosteric effector with 2,3-bisphosphoglycerate (DPG) were also examined at various levels of oxygen saturation. It was found that beta-NapP3 binds to deoxyHbA tetramer in the molar ratio of 2:1, and that one of the two beta-NapP3 competes with DPG. It was also found that beta-NapP3 binds to completely oxygenated HbA tetramer in the molar ratio of 1:1, and that 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. Furthermore, the correlations between oxygen bindings by HbA and the bindings of beta-NapP3 to HbA in the intermediate stages of deoxygenation were investigated. It was revealed that HbA as a tetramer exists in three conformational states rather than simple two states as Monod, Wyman, and Changeux had proposed.
D001665:Binding Sites; D001667:Binding, Competitive; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D010755:Organophosphates / Q000097:blood*; D009943:Organophosphorus Compounds / Q000097:blood*; D010100:Oxygen / Q000302:isolation & purification; D010108:Oxyhemoglobins / Q000378:metabolism*; D010313:Partial Pressure; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D006454:Hemoglobins; D010755:Organophosphates; D009943:Organophosphorus Compounds; D010108:Oxyhemoglobins; C011943:beta-naphthyl triphosphate; D010100:Oxygen