O'Malley S M, McDonald M J
Department of Chemistry, College of Arts and Sciences, University of Massachusetts at Lowell 01854.
J Protein Chem. 1994 Aug;13(6):561-7. doi: 10.1007/BF01901538.
A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemoglobin, in the absence and presence of human beta-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EQ) and Förster distances (R0Q) were analyzed by least-squares to determine the goodness of fit (chi R2) for the assumed distribution parameters: average distance r and half-width hw. Data for alpha-apohemoglobin in the absence and presence of beta h chains yielded values for r of 18 and 22 A and hw of 20 and 8.5 A, respectively. Although the increase in r for alpha-apohemoglobin in the presence of beta h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the alpha-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.
已采用单一能量转移距离，即唯一的内在色氨酸供体[14(A12)]与连接到唯一半胱氨酸[104(G11)]上的非荧光巯基受体探针（4-苯基偶氮苯马来酰亚胺，PAPM）之间的距离，来研究亚基组装对无血红素的人α-血红蛋白结构的影响。在5℃下于pH 7.0的0.05 M磷酸钾缓冲液中测量能量转移效率，并通过稳态溶质猝灭技术研究在不存在和存在含人β-血红素链的情况下α-脱辅基血红蛋白的结构灵活性。通过最小二乘法分析猝灭效率（EQ）和福斯特距离（R0Q），以确定假设分布参数（平均距离r和半高宽hw）的拟合优度（χR2）。不存在和存在β h链时α-脱辅基血红蛋白的数据分别得出r值为18和22 Å，hw值为20和8.5 Å。虽然存在β h链时α-脱辅基血红蛋白的r增加可能是血红素部分额外猝灭的结果，但半高宽的变化强烈表明组装蛋白内α-脱辅基血红蛋白链的灵活性降低。本文所示的结构灵活性转变可能是人类血红蛋白组装的一个重要方面。
