Fluorescence Quenching by Extrinsic Quenchers

Small molecules that act as collisional quenchers may penetrate into the internal structure of proteins, diffuse, and cause quenching upon collision with the aromatic groups. Lakowicz and Weber(53) have shown that the interaction of oxygen molecules with buried tryptophan residues in proteins leads to quenching with unexpectedly high rate constants—from 2 x 109 to 7 x 109 M l s 1. Acrylamide is also capable of quenching the fluorescence of buried tryptophan residues, as was shown for aldolase and ribonuclease 7V(54) A more hydrophobic quencher, trichloroethanol, is a considerably more efficient quencher of internal chromophore groups in proteins.(55) 

Furthermore, the quenching of internal residues in proteins by ionic quenchers, although not strong, is quite detectable.(56) A double-quenching method was developed to separate fluorescence quenching parameters characteristic of solvent-exposed and buried fluorophores.(57) The method uses two types of quenchers simultaneously, one type penetrating and the other not penetrating into the protein matrix. 

The question of what mechanisms is involved in the case of other quenchers is still unclear. For the quenching of aldolase and ribonuclease Ti by acrylamide, the activation energy is rather high, 40-45 kJ/mol,(54) but the value in the case of cod parvalbumin(60) is lower (27 kJ/mol), being similar to that for oxygen quenching. According to Bushueva et al., the efficiency of 

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