Fluorescence quenching of tryptophan by trifluoroacetamide.
Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1 X mol-1 and 1.9 X 10(9) 1 X mol-1 X s-1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555-567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1 X mol-1 and 0.92 X 10(9) 1 X mol-1 X s-1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N'-trifluoroacetyl-beta-chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.
D000081:Acetamides; D000818:Animals; D002417:Cattle; D002645:Chickens; D004529:Egg White; D005463:Fluoroacetates; D007700:Kinetics; D037102:Lectins; D009113:Muramidase; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence / Q000379:methods; D014269:Trifluoroacetic Acid; D014364:Tryptophan; D014909:Wheat Germ Agglutinins
D000081:Acetamides; D005463:Fluoroacetates; D037102:Lectins; D011506:Proteins; D014909:Wheat Germ Agglutinins; D012710:Serum Albumin, Bovine; C030688:trifluoroacetamide; D014364:Tryptophan; D014269:Trifluoroacetic Acid; D009113:Muramidase