Inhibition of NF- κB–DNA binding by mercuric ion: utility of the non-thiol reductant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), on detection of impaired NF- κB–DNA binding by thiol-directed agents

Abstract

Mercuric ion (Hg 2+), a potent thiol inhibitor, prevents expression of nuclear factor κB (NF- κB) by mercaptide bond formation with a critical cysteine moiety (cys 62) on the p50 subunit required for DNA binding. NF- κB–DNA binding is typically measured in reaction mixtures in which dithiothreitol (DTT) or other thiol reductants are used to maintain cys 62 in the reduced state. However, the presence of thiol reductants prevents accurate assessment of the Hg 2+ concentration required to prevent NF- κB–DNA binding because of competitive mercaptide bond formation. In the present studies we evaluated the efficacy of tris(2-carboxyethyl)phosphine-HCl (TCEP), a non-thiol reducing agent which does not bind Hg 2+, on NF- κB–DNA binding in vitro, using recombinant p50 protein and a 32P-labelled κB oligonucleotide. We also measured the minimal Hg 2+ concentration required to prevent this interaction in the presence of either reagent. DTT promoted NF- κB–DNA binding in concentrations from 0.25 to 2.6 m m in binding reactions. However, in the presence of 0.25 m m DTT, inhibition of NF- κB binding was seen only at Hg 2+ concentrations greater than 100 μ m and results were highly variable. In contrast, TCEP promoted NF- κB–DNA binding in a dose-related manner in concentrations from 0.25 to 6 m m. In the presence of even 6 m m TCEP, Hg 2+ prevented NF- κB–DNA binding at concentrations as low as 20 μ m in binding reactions. Similar findings were observed with regard to the thiol alkylating agent N-ethylmaleimide (NEM). These findings demonstrate the utility of TCEP as reductant in nuclear binding reaction assays involving the interaction of thiol constituents. They also demonstrate inhibition of NF- κB–DNA binding at Hg 2+ concentrations comparable to those known to initiate toxicity and apoptotic cell death in vivo.