Quenching by acrylamide and temperature of a fluorescent probe attached to the active site of ribonuclease.
The fluorescence properties of ribonuclease labelled at its active site with N-(iodoacetylamino)-ethyl-5-naphthylamine-1-sulfonic acid have been studied at different temperatures and in the presence of acrylamide. The rate constant for the quenching of the fluorescence of labelled ribonuclease by acrylamide is apparently not limited by the "accessibility" of the probe: similar values are obtained for the native and denatured states of the protein. Instead, acrylamide seems to be a rather inefficient quencher of this fluorescent group [acetamidoamino)ethyl-5-naphthylamine-1-sulfonic acid), as shown by non-linear Stern-Volmer representations, biphasic decay kinetics, and a low value of the rate constant. The fluorescence intensity of the native state of the labelled protein is highly sensitive to temperature and exhibits a 20% decrease for an increase of temperature of from 10 degrees C to 30 degrees C, independent of solvent viscosity. This thermal quenching is specific for the native conformation and disappears when the protein is unfolded. When the fluorescence lifetime of the label is shortened by addition of acrylamide, the effect of temperature becomes identical for native and unfolded structures. This suggests that the cause of the thermal quenching is the presence of conformational fluctuations within the native protein which apparently take place in the time range from 35 to 200 ns.
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology*; D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D009282:Naphthalenesulfonates; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000178:Acrylamides; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D020106:Acrylamide; C006421:1,5-I-AEDANS; D012259:Ribonuclease, Pancreatic