Collisional Quenching the Stern-Volmer Relation

Macromolecules display continuous motions. These motions can be of two main types the molecule can rotate on itself, following the precise axis of rotation, and it can have a local flexibility. Local flexibility, also called internal motions, allows different small molecules, such as solvent molecules, to diffuse along the macromolecule. This diffusion is generally dependent on the importance of the local internal dynamics. Also, the fact that solvent molecules can reach the interior hydrophobic core of macromolecules such as proteins clearly means that the term hydrophobicity should be considered as relative and not as absolute. Internal dynamics of proteins allow and facilitate a permanent contact between protein core and the solvent. Also, this internal motion permits small molecules such as oxygen to diffuse within the protein core. Since oxygen is a collisional quencher, analyzing the fluorescence data in the presence of different oxygen concentrations yields information on the internal dynamics of macromolecules. 

Dynamic quenching occurs within the fluorescence lifetime of the fluorophore, i.e., during the excited-state lifetime. This process is time-dependent. We have defined fluorescence lifetime as the time spent by the fluorophore in the excited state. Collisional quenching is a process that will depopulate the excited state in parallel to the other processes already described in the Jablonski diagram. Therefore, the excited-state fluorescence lifetime is lower in the presence of a collisional quencher than in its absence. 

Since the fluorescence intensity is proportional to the quantum yield, the Stern-Volmer equation can be written as  

A Stern-Volmer plot can be obtained also from fluorescence lifetime quenching. In fact, the fluorescence lifetime in the absence of a quencher is 

The most common quenchers are oxygen, acrylamide, iodide, and cesium ions. The kq value increases with probability of collisions between the fluorophore and quencher. Oxygen is a small and uncharged molecule, so it can diffuse easily. Therefore, the bimolecular diffusion constant kq observed for oxygen in solution is the most important between all cited quenchers. 

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