VISCOSITY DEPENDENCE OF THE SOLUTE QUENCHING OF THE TRYPTOPHANYL FLUORESCENCE OF PROTEINS
VISCOSITY DEPENDENCE OF THE SOLUTE QUENCHING OF THE TRYPTOPHANYL FLUORESCENCE OF PROTEINS
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DOI：
10.1016/0301-4622(86)80019-9
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发表时间：
1986-12-31
影响因子：
3.8
通讯作者：
HAGAMAN, KA
中科院分区：
文献类型：
--
作者：
EFTINK, MR;HAGAMAN, KA
我们已经研究了粘度依赖性的丙烯酰胺猝灭的荧光对内部色氨酸残基在鳕鱼小清蛋白和核糖核酸酶T1，以及模型系统。N-乙酰基-L-甘露酰胺和胰高血糖素。对于后者系统，丙烯酰胺猝灭的表观速率常数kq（app）显示出典型的扩散限制行为。然而，对于小清蛋白和核糖核酸酶T1，kq（app）的粘度依赖性是完全不同的。体积粘度从1增加到10 cP（通过添加甘油）时，kq（app）值几乎没有变化，但从10进一步增加到100 cP导致kq（app）显著降低。解折叠机制和淬灭剂渗透机制被认为是解释的结果。只有渗透机制被发现是一致的，我们的数据被解释为表明，淬火的限速步骤从扩散通过蛋白质基质，在低粘度，扩散通过散装溶剂，在高粘度。通过在拟合我们的数据时考虑Kramers关系，我们能够获得关于蛋白质结构中的内部波动与本体溶剂运动之间的耦合的见解。对于小清蛋白和核糖核酸酶Ti，内部动力学被发现是非常弱的耦合到散装。
We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal typtophan residues in cod parvalbumin and ribonuclease T1, as well as the model systems. N-acetyl-L-tryptophanamide and glucagon. For the latter systems, the apparent rate constant, kq(app), for acrylamide quenching shows a typical diffusion-limited behavior. For parvalbumin and ribonuclease T1, however, the viscosity dependence of kq (app) is quite different. There is little change in the kq (app) values on increasing the bulk viscosity from 1 to 10 cP (by addition of glycerol), but a further increase from 10 to 100 cP results in a significant reduction in the kq (app). Both an unfolding mechanism and a quencher penetration mechanism are considered to explain the results. Only the penetration mechanism is found to be consistent, and our data are interpreted as indicating that the rate-limiting step for quenching goes from being that of diffusion through the protein matrix, at low viscosity, to diffusion through the bulk solvent, at high viscosity. By also considering the Kramers'' relationship in fitting our data, we are able to obtain insight regarding the coupling between internal fluctuations in the structure of the protein and motion of the bulk solvent. For parvalbumin and ribonuclease Ti, the internal dynamics are found to be very weakly coupled to the bulk.