Shapiro D B, Esquerra R M, Goldbeck R A, Ballas S K, Mohandas N, Kliger D S
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
J Mol Biol. 1996 Jun 28;259(5):947-56. doi: 10.1006/jmbi.1996.0372.
Time-resolved linear dichroism (TRLD) measurements are conducted on gels of sickle cell hemoglobin following laser photolysis of the carbonyl adduct to monitor religation kinetics to hemoglobin S polymers. The return of the polymer phase to its equilibrium ligation state has been found to be about 1000 times slower than that of the solution phase hemoglobin tetramers. Several mechanisms describing this slow religation to the polymer were proposed: (1) religation occurs through a biomolecular process involving all polymer hemes, (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate, and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase. To test these mechanisms, measurements are performed on gels having different domain sizes. The results show no relation between domain size and religation kinetics. The independence of religation kinetics and domain size is most consistent with the first of the three mechanisms described above (bimolecular recombination involving all polymer hemes). This result is discussed in terms of a model in which diffusion of the ligand is inhibited in the polymer phase. An understanding of the ligand binding kinetics of sickle hemoglobin polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.
在对羰基加合物进行激光光解后，对镰状细胞血红蛋白凝胶进行时间分辨线性二色性（TRLD）测量，以监测血红蛋白S聚合物的再连接动力学。已发现聚合物相恢复到其平衡连接状态的速度比溶液相血红蛋白四聚体慢约1000倍。针对聚合物这种缓慢再连接现象，提出了几种机制：（1）再连接通过涉及所有聚合物血红素的双分子过程发生；（2）再连接通过双分子过程发生，其中只有聚合物末端的血红蛋白分子可以参与；（3）再连接通过单体相中连接的血红蛋白分子与聚合物相中未连接的血红蛋白分子的交换发生。为了验证这些机制，对具有不同结构域大小的凝胶进行了测量。结果表明结构域大小与再连接动力学之间没有关系。再连接动力学与结构域大小的独立性与上述三种机制中的第一种（涉及所有聚合物血红素的双分子重组）最为一致。根据一种模型对该结果进行了讨论，在该模型中，配体在聚合物相中的扩散受到抑制。了解镰状血红蛋白聚合物的配体结合动力学在其与红细胞循环过程中聚合物形成和熔化的相关性方面可能具有病理生理学意义。
