title: Fluorometric titration assay of affinity of tight-binding nonfluorescent inhibitor of glutathione S-transferase.
abstract: To determine inhibition constant (K(i)) of tight-binding inhibitor, the putative method estimated an apparent K(i) from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K(i), but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K(d)) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K(d) was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K(d) of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K(i) estimated via the putative method. Hence, fluorometric titration assay of K(d) of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K(i) estimated via the putative method.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
authors.forename: Bangtian
authors.initials: B
authors.lastname: Xu
authors.forename: Deng
authors.initials: D
authors.lastname: Tan
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Hu
authors.forename: Yanling
authors.initials: Y
authors.lastname: Xie
authors.forename: Jialin
authors.initials: J
authors.lastname: Qin
authors.forename: Chunyan
authors.initials: C
authors.lastname: Chen
authors.forename: Chenxiong
authors.initials: C
authors.lastname: He
authors.forename: Yuanli
authors.initials: Y
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005470:Fluorometry / Q000379:methods*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012549:Schistosoma japonicum / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D005982:Glutathione Transferase