Horiuchi K, Asai H
Eur J Biochem. 1983 Apr 5;131(3):613-8. doi: 10.1111/j.1432-1033.1983.tb07307.x.
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.
通过同时测量荧光、吸光度和氧分压，研究了荧光变构效应剂β-萘基三磷酸酯（β-NapP3）在不同氧饱和度水平下与人成人血红蛋白（HbA）的结合情况。由于先前已证明与HbA结合的β-NapP3的荧光会完全淬灭，因此可根据HbA溶液的荧光强度轻松估算与HbA结合的β-NapP3的量。还在不同氧饱和度水平下研究了上述荧光变构效应剂与2,3-二磷酸甘油酸（DPG）的交换反应。发现β-NapP3以2:1的摩尔比与脱氧HbA四聚体结合，并且两个β-NapP3中的一个与DPG竞争。还发现β-NapP3以1:1的摩尔比与完全氧合的HbA四聚体结合，并且加入DPG不会释放结合的β-NapP3。β-NapP3对DPG不结合的完全氧合HbA的非竞争性位点的结合亲和力小于对DPG也不结合的脱氧HbA的非竞争性位点的结合亲和力。此外，还研究了HbA的氧结合与β-NapP3在脱氧中间阶段与HbA结合之间的相关性。结果表明，作为四聚体的HbA存在三种构象状态，而不是像莫诺德、怀曼和尚热所提出的那样简单的两种状态。
