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

Mercuric ion (Hg2+), a potent thiol inhibitor, prevents expression of nucle ar 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 dithioth reitol (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-t hiol reducing agent which does oat bind Hg2+ on NF-kappa B-DNA binding in v itro, using recombinant p50 protein and P-32-labelled kappa B oligonucleoti de. We also measured the minimal Hg2+ concentration required to pre cent th is interaction in the presence of either reagent, DTT promoted NF-kappa B-D NA binding in concentrations from 0.25 to 2.6 mM in binding reactions. Howe ver, 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 hig hly variable. In contrast, TCEP promoted NF-kappa B-DNA binding in a dose-r elated 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 t o the thiol alkylating agent N-ethylmaleimide (NEM). These findings demonst rate the utility of TCEP as reductant in nuclear binding reaction assays in volving the interaction of thiol constituents. They also demonstrate inhibi tion of NF-kappa B-DNA binding at Hg2+ concentrations comparable to those k nown to initiate toxicity and apoptotic cell death irt vivo. (C) 2000 Elsev ier Science Ltd. All rights reserved.