Figure 6

Equilibrium binding of NADH to Mt CS assessed by monitoring the protein fluorescence quench upon binary complex formation. The binding of NADH to Mt CS causes a quench in protein fluorescence (λ_exc = 290 nm; 310 ≤ λ_em ≤ 510 nm; with a maximum at 345.5 nm). The Mt CS enzyme solution (1 μM) was titrated with increasing concentrations of NADH, and the data points (fluorescence intensities at 345.5 nm) were fitted to a hyperbolic equation (solid line). Inset: Emission spectra of free Mt CS (1 μM) and enzyme in the presence of 80 μM NADH. Emission spectrum of free enzyme shows a peak at 345.5 nm and no emission at ~450 nm. Emission spectrum of Mt CS in the presence of NADH shows a quench in protein fluorescence concomitant to an increase in nucleotide fluorescence at ~450 nm.