Aggregation of pyrene-labeled microsomal glutathione S-transferase. Effect of concentration.

Abstract

Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyrenyl)maleimide which modified 1 mol thiol residue/mol protein. The enzyme activity increased about tenfold after the binding. The pyrene-labeled microsomal glutathione S-transferase exhibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S-transferase, to larger aggregates. At 25 degrees C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 microM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N-(1-pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.