Measurement of NO Production by eNOS

The release of NO generated by eNOS was measured by the oxyhemoglobin to methemoglobin conversion assay following the procedures developed by Sheta et al. and Stuehr et al. with minor modification (33 (link),34 (link)). The reaction system contained 5 μM oxyhemoglobin, 4 μg purified human eNOS, 0.2 mM DTT, 1 mM CaCl₂, 1 μg/ml CaM, 5 μM BH₄, 200 μM NADPH, in 50 mM HEPES buffer, pH 7.4, in a total volume of 100 μl. The reaction was initiated by the addition of 100 μM L-arginine. NO generation was measured by monitoring the change in absorbance A₄₀₁-A₄₁₀, and a millimolar absorbance coefficient of 38 mM^-1cm^-1. For activity measurements of the ONOO⁻-treated eNOS, the appropriate volume of diluted ONOO⁻ was added and the samples were incubated on ice for 10 minutes. Since the half-life of ONOO⁻ at neutral pH values is about 1 second, it is completely decomposed prior to addition to the assay mixture (35 (link)). For activity measurements with TPEN, a 1.25 mM TPEN solution was prepared in water, and this solution was used for the preparation of the reaction buffer above such that the final TPEN concentration was 1 mM.