Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates.
Molecules of muscle aldolase A exposed to acrylamide change their conformation via I1, T, I2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1. 8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0. 2-0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules.
Dobryszycki
P
P
Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wybrzeze Wyspiańskiego 27, 50-370, Wroclaw, Poland.
Rymarczuk
M
M
Gapiński
J
J
Kochman
M
M
D020106:Acrylamide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D008027:Light; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014364:Tryptophan / Q000032:analysis
D013438:Sulfhydryl Compounds; D020106:Acrylamide; D014364:Tryptophan; D005634:Fructose-Bisphosphate Aldolase