Acrylamide quenching of the fluorescence of glyceraldehyde-3-phosphate dehydrogenase: reversible and irreversible effects.
The acrylamide quenching of the tryptophan fluorescence of apo and holo glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was studied. In the case of apo-GAPDH, the steady state fluorescence quenching cannot be described by the classical Stern-Volmer equation: strong cooperative quenching is observed. In the presence of Pi and/or cofactor NAD+, an inaccessible fraction appears. Cooperative quenching is partially suppressed in the presence of Pi and fully absent in the presence of NAD+. The measurements of the fluorescence lifetimes of the holo-enzyme by phasefluorometry allow the resolution of two lifetimes. The long-lived component is quenched by acrylamide, the short-lived component is not. Quenching induces a red shift of the steady state emission peak. The quenching parameters from the lifetime measurements allow the quantitative description of the steady state fluorescence quenching data. In agreement with the observations of Orstan and Gafni (Photochemistry and Photobiology, (1990) 31, 725-731), we find that acrylamide causes a slow, irreversible loss of activity and a reduction of titratable thiol groups when it acts on the apo-enzyme. This inactivation is strongly reduced in the presence of NAD+. We show that this inactivation is also slowed down by the presence of Pi, and that it is accompanied by a loss of the NAD+ binding site. Blocking the thiol groups with 5,5'-dithio-bis-(2-nitrobenzoic acid) does not lead to a protection against the irreversible inactivation by acrylamide, showing that reactions other than thiol modifications are involved in the irreversible effect. A fraction of the inactivation can be reversed by treatment with mercapto-ethanol.
Bastyns
K
K
Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit Leuven, Belgium.
Engelborghs
Y
Y
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D001051:Apoenzymes; D020106:Acrylamide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases