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.

The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase, butyrylcholinesterase, glutathione S-transferase, lactoperoxidase, and carbonic anhydrase isoenzymes I, II, IX, and XII

“…GST was effectively inhibited by CAPE, with a K i value of 0.453 nM (Table 1). It was reported that the inhibitor of GST-bearing suitable linkers could concomitantly bind to two active sites of GST and usually possess K i values at nanomolar levels and excellent enzyme-selectivity 138 .…”

Achievement of linear response for competitive bioaffinity assays of ligands: criteria of optimized interaction systems

“…On the other hand, eqn (11) always applies for the same C PF under competitive binding in the same interaction system; thus, eqn (12) applies when C XF /C XT < 10% is validated. With C PF negligible to C XT for the desired LOQ, the difference between C PT and C RBU is an approximate of C XBU ; therefore, eqn (13) applies.…”

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers

“…The fluorescence of the tryptophan residues in GST at 340 nm under excitation at 280 nm was susceptible to the binding of ligands. Binding ratios of the divalent conjugate from BDEA plus excess GSH to GSTM and GSTA were thus estimated via fluorometric titration 36 . GSH, BDEA and their conjugate had negligible fluorescence signals at 340 nm under excitation at 280 nm.…”

“…The inhibition constant ( K i ) of the divalent conjugate of BDEA to GSTM was estimated. After pre-incubation of the purified divalent conjugate with GSTM alone for 10–30 min to achieve divalent binding, the analysis of the responses of residual activities of GSTM to the total concentrations of the divalent conjugate, according to a model for a slow tight-binding inhibitor (Methods Equation (1)) 27 , 36 , gave an apparent K i of (0.48 ± 0.04) nM ( n = 3). The divalent conjugate of BDEA and GSH was thus a slow tight-binding inhibitor to GSTM with the highest affinity in searchable data 8 , 10 , 17 , 22 , 24–27 , 29 , 31 .…”

“…To approximate K i for the divalent GSH conjugate of BDEA as a tight binding inhibitor, Eq. (1) considering the binding sites depletion in GST to develop the equilibrium of the binding reaction between active sites of GST and GSH conjugate was fit to the response curve 27 , 36 , 37 : …”