Eftink M R, Hagaman K A
Biophys Chem. 1986 Dec 31;25(3):277-82. doi: 10.1016/0301-4622(86)80019-9.
We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal tryptophan 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 T1, the internal dynamics are found to be very weakly coupled to the bulk.
我们研究了丙烯酰胺对鳕鱼小清蛋白和核糖核酸酶T1内部色氨酸残基荧光猝灭的粘度依赖性，以及模型体系N-乙酰-L-色氨酸酰胺和胰高血糖素的情况。对于后一种体系，丙烯酰胺猝灭的表观速率常数kq(app)呈现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)的粘度依赖性却大不相同。将本体粘度从1 cP增加到10 cP（通过添加甘油）时，kq(app)值变化不大，但从10 cP进一步增加到100 cP会导致kq(app)显著降低。我们考虑了展开机制和猝灭剂渗透机制来解释这些结果。结果发现只有渗透机制是一致的，我们的数据被解释为表明猝灭的限速步骤从低粘度下通过蛋白质基质的扩散转变为高粘度下通过本体溶剂的扩散。通过在拟合数据时考虑克拉默斯关系，我们能够深入了解蛋白质结构内部波动与本体溶剂运动之间的耦合。对于小清蛋白和核糖核酸酶T1，发现其内部动力学与本体的耦合非常弱。
