Add time:07/22/2019         Source:sciencedirect.com

Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which is less reactive, favoring the enzyme-catalyzed reaction. 6-Chloroacetyl-2-dimethylaminonaphthalene was found to be a substrate for all GSTs tested. Apparent kcat/Km values (ranging between 10 and 500 mM−1s−1) revealed a strong preference for soluble GSTP1-1, GSTA1-1, and activated MGST1. Thus, 6-chloroacetyl-2-dimethylaminonaphthalene can be used in a highly sensitive assay of these GSTs. 6-Acetyl-2-dimethylaminonaphthalene derivatives are very sensitive toward solvent polarity and potentially also toward properties of binding sites in proteins. Upon binding of the conjugate to GSTs the fluorescence intensity decreased and the emission maximum was blue-shifted. Therefore the interaction of the conjugate with GSTs can be characterized with regard to both binding affinity and kinetics by stopped-flow measurements, and 6-chloroacetyl-2-dimethylaminonaphthalene can be a valuable aid in mechanistic investigations of GSTs, especially those which possess low intrinsic fluorescence.

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