Somogyi B, Punyiczki M, Hedstrom J, Norman J A, Prendergast F G, Rosenberg A
Department of Biophysics, University Medical School of Pecs, Hungary.
Biochim Biophys Acta. 1994 Nov 16;1209(1):61-8. doi: 10.1016/0167-4838(94)90137-6.
Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.
我们最近的平衡透析研究表明，蛋白质能够优先与丙烯酰胺相互作用（普尼茨基等人，《生物物理化学》，1993年，第47卷，第9 - 19页）。蛋白质体积中存在相当数量的丙烯酰胺（尽管结合较弱），再加上简单的猝灭门控模型无法解释核糖核酸酶T1（RNase T1）中色氨酸（Trp）荧光猝灭的粘度依赖性，这促使我们探索一种新模型，即猝灭的两相模型。根据该模型，动态猝灭是由激发瞬间已存在于蛋白质相中的猝灭剂分子完成的。此时，一些分子可能与荧光团形成遭遇复合物，从而导致观察到的静态贡献。我们使用从我们的模型推导出来的速率方程来研究丙烯酰胺对RNase T1中Trp荧光猝灭的粘度依赖性。该模型使我们能够分离共溶剂效应：对蛋白质以及猝灭剂分子在本体相和蛋白质相之间分布的化学效应，以及由于本体粘度与影响蛋白质基质分子内波动的局部摩擦之间的耦合而产生的粘度效应。我们用本体粘度η和耦合常数κ来表示局部摩擦（摩擦 = ηκ）。添加高达65%的甘油时，κ的值为0.50。表观双分子猝灭常数的粘度依赖性是两种补偿效应的组合：化学活性的变化和结构波动模式的变化。
