Acrylamide fluorescence quenching

The Dp and Dq are the diffusion coefficients of probe and quencher, respectively, N is the number molecules per millimole, andp is a factor that is related to the probability of each collision causing quenching and to the radius of interaction of probe and quencher. A more detailed treatment of fluorescence quenching including multiexponential intensity decays and static quenching is given elsewhere/635 There are many known collisional quenchers (analytes) which alter the fluorescence intensity and decay time. These include O2/19 2( 29 64 66) halides,(67 69) chlorinated hydrocarbons/705 iodide/715 bromate/725 xenon/735 acrylamide/745 succinimide/755 sulfur dioxide/765 and halothane/775 to name a few. 

A. Follenius and D. Gerard, Acrylamide fluorescence quenching applied to tyrosyl residues in proteins, Photochem. Photobiol. 38, 373-376 (1983). 

M. R. Eftink and L. A. Selvidge, Fluorescence quenching of liver alcohol dehydrogenase by acrylamide, Biochemistry 21, 117-125 (1982). 

Fluorescence quenching has proven to be a powerful means to determine location of tryptophans. Small organic molecules, such as acetone, acrylamide, and amino acids, have been used to quench fluorescence of tryptophans which are exposed to the solvent.(50 51) These molecules apparently quench by close interaction and so provide a tool to determine the surface accessibility7 of tryptophan in a protein. 

Ribonuclease T and Alcohol Dehydrogenase Fluorescence Quenching by Acrylamide 128... 

The constant K is known as the Stem-Volmer quenching constant /cQ is the rate constant for the quenching reaction, and t0 the lifetime in the absence of quencher. Fluorescence quenching of tryptophan in proteins by acrylamide or 02 has been used to determine whether tryptophan side chains are accessible to solvent or are "buried" in the protein.141 142 The long-lived phosphorescence of tryptophan can be studied in a similar... 

Other measures of protein flexibility have been found to correlate with thermal stability. One is resistance to proteolysis (Daniel et al., 1982 Fontana, 1988). Another is the quenching of buried tryptophan fluorescence by acrylamide, used in a study by Varley and Pain (1991). Both these processes are mediated by the same combination of local and global unfolding events that determine rates of hydrogen exchange. Their rates will depend on the ability of another molecule, acrylamide or a proteolytic enzyme, to penetrate into normally buried regions of the protein in order to either quench fluorescence or cleave peptide bonds. 

The efficiency and mechanism of fluorescence quenching by acrylamide and succinimide for simple aromatic fluorophores has... 

Acrylamide and Oj fluorescence quenching has been used as a probe of solvent accessibility of aromatic fluorophores complexed... 

Once again melittin illustrates the effect of protein structure on the fluorescence emission. Acrylamide quenching data for melittin monomer and tetramer are shown in Fig. 8. Stem-Volmer plots are often used to present quenching data. The Stem-Volmer equation is... 

Glucose Glucoseoxidase + Ru complexes/ acrylamide polymer Fluorescence quenching of O2... 

Figure 13 A modified Stem-Voimer plot for the fluorescence quenching of HDLi by iodide in the presence of varying amounts of acrylamide. The data are shown for HDL. samples obtained from rats fed with manganese-deficient (top) and manganese-adequate (bottom) diet. The fluorescence was monitored at 338 nm (Igxc = 295 nm) characteristic of tryptophan residues. Reproduced with permission from Taylor PN, Patterson HH and Ktimis-Tavatzis DJ (1997) A fluorescence double-quenching study of native lipoproteins in an animal model of manganese deficiency. Biological Trace Element Research 60 69-80.

In the absence of alternative 3-D structural information, fluorescence quenching can be used to study the structural and functional dynamics of a protein. This method is based on the reduction of fluorescence efficiency of Trp, Tyr, or an artificially introduced fluorophor in the presence of substrates (analogs), inhibitors, or small molecules (e.g., iodide ion and acrylamide). 

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)... 

P. C. Leavis, E. Gowell, and T. Tao, Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits, Biochemistry 23, 4156 1161 (1984). 

The anisotropy decay of the tryptophan fluorescence of both model peptides and biologically active peptides containing a single tryptophan residue has been determined in various studies. Even in the case of the tripeptide H-Gly-Trp-Gly-OH quenched by acrylamide the anisotropy decay displayed two correlation times with values of 39 and 135 ps. 44 The shorter correlation time was thought to be due to motions of the indole ring relative to the tripeptide. In the case of ACTH(l-24) the fluorescence anisotropy decay of the single tryptophan residue in position 9 of the peptide sequence obtained in phosphate buffer (pH 7, 3.5 °C) was also double-exponential. 29 The shorter rotational correlation time (0 = 92ps)... 

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