title: Synthesis and characterization of a series of highly fluorogenic substrates for glutathione transferases, a general strategy.
abstract: Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.
authors.affiliation: Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Aya
authors.initials: A
authors.lastname: Shibata
authors.forename: Mika
authors.initials: M
authors.lastname: Ito
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shuto
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ito
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Abe
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D005982:Glutathione Transferase