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. 2014 Feb 11;53(8):1284–1295. doi: 10.1021/bi401307r

Figure 7.

Figure 7

Kinetics of tight binding site formation for ECo-nNOS. Shown is the effect of the time interval between BH4 and 4-amino-BH4 administration on the rate of NO formation by ECo-nNOS, as measured with the oxyHb assay. Closed circles: the enzyme was preincubated with 1 μM 4-amino-BH4 for the indicated time, after which the activity was determined in the presence of 10 μM BH4. The curve through the data points is the best fit to a single exponential with parameters: kobs = 1.6 ± 0.3 min–1, Act0 = 151 ± 4 nmol·mg–1·min–1, Act∞ = 87 ± 2 nmol·mg–1·min–1. Open circles: the enzyme was preincubated with 1 μM BH4 for the indicated time, after which the activity was determined in the presence of 10 μM 4-amino-BH4. The curve through the data points is the best fit to a single exponential with parameters: kobs = 0.23 ± 0.10 min–1, Act0 = 16 ± 4 nmol·mg–1·min–1, Act∞ = 79 ± 11 nmol·mg–1·min–1. In both cases data points are shown ± SEM (n = 3). Preincubation conditions: 2 μg/mL (∼11 nM) ECo-nNOS, 0.2 mM Arg, 0.2 mM NADPH, 5 μM FAD, 5 μM FMN, 0.5 mM CaCl2, 5 μM oxyHb, 1000 U/mL SOD, 50 mU/mL CAT, 0.2 mM CHAPS, 0.1 mM EDTA, 50 mM TEA (pH 7.4), and 1 μM BH4 or 4-amino-BH4 at 4 °C. The assay conditions were the same except for the temperature (37 °C), the presence of 10 μM 4-amino-BH4 or BH4 (in the case of preincubation with BH4 and 4-amino-BH4, respectively), and the presence of 10 μg/mL CaM, which was added to start the reaction. In both cases, data points are shown ± SEM (n = 3).