| Assay Method Information | |
| | Hemoglobin Capture Assay |
| Description: | The assay was performed at 37 °C in HEPES buffer (100 mM, with 10% glycerol, pH 7.4) in the presence of 10 uM L-arginine. Also included were 100 uM NADPH, 0.83 mM CaCl2, approximately 320 units/mL calmodulin, 10 uM tetrahydrobiopterin, andhuman oxyhemoglobin (3 uM). This assay was performed in 96-well plates using a Biotek Gen5 microplate reader. NO production was read by monitoring the absorbance at 401 nm (resulting from the conversion of oxyhemoglobin to methemoglobin). Kinetic readouts were recorded for 6 min. Each compound was assayed in at least duplicate, and seven to nine concentrations (from 50 nM to 200 uM) were used to construct dose-response curves. IC50 values were calculated by nonlinear regression using GraphPad Prism, and Ki values were obtained using the Cheng-Prusoff equation [Ki = IC50/(1 + [S]/Km)] with the following Km values: 1.3 uM for rat nNOS, 1.7 uM for the rat nNOS D597N mutant, and 1.9 uM for the rat nNOS. |
| Affinity data for this assay | |
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