Demehin Afolorunso Andrew, Abugo Omoefe O, Jayakumar Rajadas, Lakowicz Joseph R, Rifkind Joseph M
Molecular Dynamics Section, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Biochemistry. 2002 Jul 9;41(27):8630-7. doi: 10.1021/bi012007e.
We have studied the binding of hemoglobin to the red cell membrane by centrifugation and fluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specifically reacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band 3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobin bound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding was quantitatively compared with the binding determined by centrifugation, which measures binding to band 3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 association constants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpreted in terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interaction associated with the conformational change produced when hemoglobin binds results in a 2.8-fold decrease in the intrinsic constant of (1.54 +/- 0.25) x 10(7) M(-1) for the binding of the second hemoglobin molecule. From the changes in lifetime produced by binding the first and second hemoglobin molecules, it was possible to show that the conformational change associated with binding the second hemoglobin molecule results in a decrease of the heme-eosin distance from 47.90 to 44.78 A. Reaction of cyanate with the alpha-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding of hemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobin molecule to band 3 decreases by a factor of 29 to (5.34 +/- 0.15) x 10(5) M(-1). The anticooperative interaction is greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobin tetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobin is very different with the second HbOCN increasing the heme-eosin distance to 55.99 A. The utilization of eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directly study the binding to band 3. At the same time a sensitive probe of the conformational changes, which occur when hemoglobin binds to band 3, is provided.
我们通过离心和荧光方法研究了血红蛋白与红细胞膜的结合。完整的红细胞用嗜酸性-5-马来酰亚胺（EM）标记，它与带3的赖氨酸430特异性反应。尽管该残基不是与血红蛋白结合相关的带3细胞质结构域（cdb3）的一部分，但当血红蛋白与内翻囊泡（IOV）结合时仍观察到荧光猝灭。将用于测量带3结合的荧光猝灭与通过离心测定的结合进行了定量比较，离心测量的是与带3和非带3位点的结合。对于离心，有必要纳入从胰凝乳蛋白酶处理的IOV确定的非带3缔合常数。血红蛋白与带3的结合被解释为两个血红蛋白四聚体与每个带3二聚体的结合。与血红蛋白结合时产生的构象变化相关的反协同相互作用导致第二个血红蛋白分子结合的内在常数（1.54±0.25）×10⁷ M⁻¹下降了2.8倍。根据结合第一个和第二个血红蛋白分子产生的寿命变化，有可能表明与结合第二个血红蛋白分子相关的构象变化导致血红素 - 嗜酸性距离从47.90 Å减小到44.78 Å。氰酸盐与血红蛋白的α-氨基反应（HbOCN）显示出会使血红蛋白与带3和非带3位点的结合大幅下降。第一个血红蛋白分子与带3结合的内在常数下降了29倍，降至（5.34±0.15）×10⁵ M⁻¹。第二个血红蛋白四聚体与带3结合时的反协同相互作用更大，内在常数下降了3.8倍。此外，结合血红蛋白产生的构象变化的性质非常不同，第二个HbOCN使血红素 - 嗜酸性距离增加到55.99 Å。利用嗜酸性-5-马来酰亚胺反应的红细胞膜来研究血红蛋白结合使得直接研究与带3的结合成为可能。同时，它为血红蛋白与带3结合时发生的构象变化提供了一个灵敏的探针。
