Figure 4
Effect of UDP-GlcNAC on the intrinsic tryptophan fluorescence of CHS3a-SGC-423 protein and Binding Profiles. (A) Emission spectra of 1 μM CHS3a-SGC-423 incubated in 20 mM potassium phosphate, pH 6.5, 10 mM MgOAc with increasing concentrations of UDP-GlcNAC (0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, and 8 mmol.L-1) from the top to the bottom (continuous lines). Reference spectrum for CHS3a-SGC-423 without substrate shown with a dotted line. Maximal fluorescence value « F » at 340 nm was used to quantify bound proteins, « F0 » corresponds to the unbound protein signal. (B) Saturation curve [Ebound]/[Etotal] = f([UDP-GlcNAc]) presents an hyperbolic profile. [Ebound]/[Etotal] was obtained from (1 - F/F0) point at 340 nm for each curve in panel A. (C) Scatchard plot of UDP-GlcNAc specific binding to CHS3a-SGC-423. [Ebound]/([S]*[Etotal]) mmol-1.L versus [Ebound]/[Etotal] was plotted.