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. 2009 Jul 11;284(36):24354–24362. doi: 10.1074/jbc.M109.019919

FIGURE 1.

FIGURE 1.

The effect of C3 on the fluorescence of GSNOR bound NADH in the presence or absence of 12-HDDA. A, changes in the fluorescence of NADH (curve a) upon the sequential addition of GSNOR (curve b) and C3 (curve c). To a solution of 1.7 μm NADH were added 2 μm GSNOR and 50 μm C3 in sequence, and the fluorescence of the solution was measured each time (λex = 350 nm; λem = 375–550 nm). B, changes in the fluorescence of NADH (curve a) upon the sequential addition of GSNOR (curve b), 12-HDDA (curve c), and C3 (curve d). To a solution of 1.7 μm NADH were added 2 μm GSNOR, 810 μm 12-HDDA, and 50 μm C3 in sequence, and the fluorescence was measured each time (λex = 350 nm; λem = 375–550 nm). C, binding of C3 to GSNOR·NADH complex. The change in fluorescence of a 1.7 μm NADH and 2 μm GSNOR mixture (λex = 350 nm; λem = 455 nm) with increasing concentrations of C3 was fitted to a single-site binding model (Equation 1) using GraphPad Prism 4. The equilibrium dissociation constant of C3 was 1.9 ± 0.14 μm. All of the fluorescence studies were conducted at room temperature in 50 mm potassium phosphate, pH 7.5.