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 °C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 μM. 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.
|Number of pages||3|
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1993|
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