Lakowicz J R, Zelent B, Gryczynski I, Kuśba J, Johnson M L
Department of Biological Chemistry, University of Maryland at Baltimore, School of Medicine 21201.
Photochem Photobiol. 1994 Sep;60(3):205-14. doi: 10.1111/j.1751-1097.1994.tb05092.x.
We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N-acetyl-L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20 degrees C. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NATA by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.
我们使用GHz频域荧光法研究了在20℃下丙二醇中N-乙酰-L-色氨酸酰胺（NATA）被丙烯酰胺碰撞猝灭时随时间变化的强度衰减。在丙烯酰胺存在下，NATA的强度衰减变得越来越不均匀。NATA的强度衰减与用于猝灭的Collins-Kimball辐射边界条件（RBC）模型不一致。稳态Stern-Volmer图显示出明显的向上弯曲。在玻璃化丙二醇（-60%）的低温下，在激发态寿命期间不会发生平移扩散，观察到丙烯酰胺对NATA的猝灭。Smoluchowski和RBC猝灭模型预测在没有平移扩散的情况下不会发生任何猝灭。因此，这些频域和稳态数据表明NATA和丙烯酰胺之间存在通过空间的猝灭相互作用。丙烯酰胺对NATA的猝灭速率似乎指数依赖于荧光团-猝灭剂的分离距离。时间分辨和稳态数据的比较提供了一种灵敏的方法来确定荧光团-猝灭剂相互作用的距离依赖性。距离依赖性猝灭速率也解释了Stern-Volmer图的向上弯曲，这在丙烯酰胺猝灭中经常观察到。这些结果表明，在解释丙烯酰胺对蛋白质的猝灭数据时需要考虑距离依赖性猝灭速率。
