Lakowicz J R, Joshi N B, Johnson M L, Szmacinski H, Gryczynski I
J Biol Chem. 1987 Aug 15;262(23):10907-10.
We used 2-GHz frequency-domain fluorometry to examine the intensity decays of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease in the presence and absence of quenching by oxygen or acrylamide. When analyzed with a multiexponential model, the decays of NATA and nuclease both become more heterogeneous in the presence of quenching. We attribute the increased complexity to transient effects in quenching or equivalently a time-dependent rate constant for quenching. The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t)) and the radiation model, which is known to correct some flaws in the more approximate Smoluchowski model. The radiation model provides improved fits to the data, as evidenced by average 10-fold decreases in chi R2. The radiation model also provides an estimate of the sum of the diffusion coefficients and the specific rate constant for quenching. The apparent diffusion coefficients for acrylamide and oxygen in nuclease, as seen by its single tryptophan (residue 140) are 15- and 11-fold lower than in water, respectively. The apparent values of the oxygen diffusion coefficient in water, as seen by NATA, are 2- to 3-fold larger than expected from earlier steady-state measurements. The ability to recover the detailed form of the intensity decays by the frequency-domain method should allow comparison of experimental results with calculated trajectories of quenchers in proteins.
我们使用2-GHz频域荧光法，在有或无氧气或丙烯酰胺淬灭的情况下，检测了N-乙酰-L-色胺（NATA）和蛋白质葡萄球菌核酸酶的强度衰减。当用多指数模型分析时，在淬灭存在的情况下，NATA和核酸酶的衰减都变得更加不均匀。我们将这种增加的复杂性归因于淬灭中的瞬态效应，或者等效地归因于淬灭的时间依赖性速率常数。使用Smoluchowski模型（exp(-t/tau-2b√t)）和辐射模型对频域数据进行了分析，已知辐射模型可以纠正更近似的Smoluchowski模型中的一些缺陷。辐射模型对数据的拟合得到了改善，χR2平均下降了10倍就证明了这一点。辐射模型还提供了扩散系数之和以及淬灭的特定速率常数的估计值。从核酸酶的单个色氨酸（残基140）来看，丙烯酰胺和氧气在核酸酶中的表观扩散系数分别比在水中低15倍和11倍。从NATA来看，水中氧气扩散系数的表观值比早期稳态测量预期的大2至3倍。通过频域方法恢复强度衰减详细形式的能力，应该能够将实验结果与蛋白质中淬灭剂的计算轨迹进行比较。
