Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite
J. Clin. Invest. Ming-Hui Zou, et al. 109:817 doi:10.1172/JCI14442 [Go to this article.]

Figure 3
Comparison of ONOO^– reactivity with the zinc-thiolate cluster of recombinant eNOS and its cofactor, BH₄. (a) BH₄ is oxidized by ONOO^– but not by tetranitromethane (TNM). Note that 50 μmol/l ONOO^–, which nearly completely dissociated eNOS dimers in low-temperature SDS-PAGE under reducing conditions, did not cause detectable oxidation of BH₄. These results represent 15 assays in three independent experiments. (b) Effects of BH₄ and/or L-arginine on ONOO^–-induced dissociation of eNOS. BH₄ (100 μmmol/l) and L-arginine (1 mmol/l) were added to purified eNOS 10 minutes before addition of ONOO^–. Blot shown is representative of three independent experiments. (c) Representative blot of three independent experiments showing that TNM, which did not oxidize BH₄, results in dissociation of eNOS dimer into monomer under reducing conditions. Purified eNOS was exposed to TNM (0.1 or 1 mmol/l) or vehicle containing DMSO at room temperature for 30 minutes. eNOS dimer and monomer were assayed by low-temperature SDS-PAGE and visualized by Coomassie staining.