title: Effect of acrylamide on aldolase structure. I. Induction of intermediate states.
abstract: Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.
authors.affiliation: Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wybrzeze Wyspiańskiego 27, 50-370, Wroclaw, Poland.
authors.forename: P
authors.initials: P
authors.lastname: Dobryszycki
authors.forename: M
authors.initials: M
authors.lastname: Rymarczuk
authors.forename: G
authors.initials: G
authors.lastname: Bułaj
authors.forename: M
authors.initials: M
authors.lastname: Kochman
mesh_terms: D020106:Acrylamide / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
chemical_list: D020106:Acrylamide; D014364:Tryptophan; D005634:Fructose-Bisphosphate Aldolase