Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.
Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.
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DOI：
10.1021/bi00491a013
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发表时间：
1990-10
期刊：
影响因子：
2.9
通讯作者：
Serge Pin;Catherine A. Royer;Enrico Gratton;Bernard Alpert;Gregorio Weber
中科院分区：
文献类型：
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作者：
Serge Pin;Catherine A. Royer;Enrico Gratton;Bernard Alpert;Gregorio Weber
采用稳态荧光各向异性、多频相荧光法和高静水压力法研究了成人氧合血红蛋白亚基的解离。用外源性荧光探针5-（二甲氨基）萘-1-磺酰氯（DNS-Cl）标记通过两种纯化程序（离心大量制备或阴离子交换色谱进一步分离）获得的人血红蛋白。该探针的长荧光寿命允许观察大分子翻滚，从而提供了一种在不同质子和有机磷酸盐浓度的溶液条件下观察亚基解离后配合物大小变化的方法。在pH为7时，大量和分离血红蛋白的丹基化制剂显示出各向异性的浓度依赖性降低，尽管不相同，但各向异性的降低只能由四聚体到二聚体的解离引起。我们主要观察到了pH值为9的二聚体，以及在饱和六磷酸肌醇（IHP）存在下四聚体的小不稳定性。高压实验允许观察血红蛋白二聚体解离成单体。从这些测量中，我们估计二聚体解离常数在0.1和1nm之间。我们比较了目前从稳态和时间分辨荧光数据中获得的血红蛋白亚基亲和力的结果与以前通过凝胶过滤、沉淀和动力学技术获得的结果。这些比较表明血红蛋白制剂中存在一定程度的构象异质性。
The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.