Principles of Fluorescence Spectroscopy

pp 277-330

Quenching of Fluorescence

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Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and colli-sional quenching. In this chapter we will be concerned primarily with quenching resulting from collisional encounters between the fluorophore and quencher, which is called collisional or dynamic quenching. We will also discuss static quenching, which can be a valuable source of information about binding between the fluorescent sample and the quencher. Static quenching can also be a complicating factor in the data analysis. In addition to the processes described above, apparent quenching can occur due to the optical properties of the sample. High optical densities or turbidity can result in decreased fluorescence intensities. This trivial type of quenching contains little molecular information. Throughout this chapter we will assume that such trivial effects are not the cause of the decreases in fluorescence intensity.

Fluorescence quenching has been widely studied both as a fundamental phenomenon, and as a source of information about biochemical systems. These biochemical applications of quenching are due to the molecular interactions that result in quenching. Both static and dynamic quenching require molecular contact between the fluorophore and quencher. In the case of collisional quenching, the quencher must diffuse to the fluorophore during the lifetime of the excited state. Upon contact, the fluorophore returns to the ground state, without emission of a photon. In general, quenching occurs without any permanent change in the molecules, that is, without a photochemical reaction. In static quenching a complex is formed between the fluo-rophore and the quencher, and this complex is nonfluores-cent. For either static or dynamic quenching to occur the fluorophore and quencher must be in contact. The requirement of molecular contact for quenching results in the numerous applications of quenching. For example, quenching measurements can reveal the accessibility of fluo-rophores to quenchers. Consider a fluorophore bound either to a protein or a membrane. If the protein or membrane is impermeable to the quencher, and the fluorophore is located in the interior of the macromolecule, then neither colli-sional nor static quenching can occur. For this reason quenching studies can be used to reveal the localization of fluorophores in proteins and membranes, and their permeabilities to quenchers. Additionally, the rate of collisional quenching can be used to determine the diffusion coefficient of the quencher.