Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates

Molecules of muscle aldolase A exposed to acrylamide change their conformat ion via I-1, T, I-2, D intermediates [1] and undergo a slow irreversible ch emical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two clas ses of Trp residues of 1.8 ns and 4.9 ns fluorescence lifetime have been fo und. Acrylamide (0.2-0.5 M) increases lifetime of longer-lived component, y et the transfer of aldolase molecules even from higher (1.0 M) perturbant c oncentration to a buffer, allows regain original Trp fluorescence lifetime. I-1, detected at about 0.2 M acrylamide, represents low populated tetramer s of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activit y. These molecules are able to exchange their subunits with aldolase C in o pposition to the native molecules. At transition point for It appearance (1 .8 M acrylamide), aldolase becomes highly unstable: part of molecules disso ciate into subunits which in the absence of perturbant are able to reassoci ate into active tetramers, the remaining part undergoes irreversible denatu ration and aggregation. Some expansion of aldolase tetramers takes place pr ior to dissociation. D, observed above 3.0 M acrylamide, consists of irreve rsibly denatured enzyme molecules. (C) 1999 Elsevier Science B.V. All right s reserved.