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

Mercuric ion (Hg2+), a potent thiol inhibitor, prevents expression of nuclear factor kappa B (NF-kappa B) by mercaptide bond formation with a critical cysteine moiety (cys(62)) on the p50 subunit required for DNA binding, NF-KB-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 Hg2+ concentration required to prevent NF-KB-DNA binding because of competitive mercaptide bond formation. In the present studies we evaluated the efficacy of tris(2-carboxyethyl)phoshine-HCl (TCEP), a non-thiol reducing agent which does oat bind Hg2+ on NF-kappa B-DNA binding in vitro, using recombinant p50 protein and P-32-labelled kappa B oligonucleotide. We also measured the minimal Hg2+ concentration required to pre cent this interaction in the presence of either reagent, DTT promoted NF-kappa B-DNA binding in concentrations from 0.25 to 2.6 mM in binding reactions. However, in the presence of 0.25 mil DTT, inhibition of NF-kappa B binding was seen only at Hg2+ concentrations greater than 100 mu M and results were highly variable. In contrast, TCEP promoted NF-kappa B-DNA binding in a dose-related manner in concentrations from 0.25 to 6 mM. In the presence of even 6mM TCEP, Hg2+ prevented NF-kappa B-DNA binding at concentrations as low as 20 mu 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-kappa B-DNA binding at Hg2+ concentrations comparable to those known to initiate toxicity and apoptotic cell death irt vivo. (C) 2000 Elsevier Science Ltd. All rights reserved.