| Assay Method Information | |
| | Fluorescence Binding Assay |
| Description: | Fluorescence spectroscopy was used to determine the G6P and F420 dissociationconstants (Kd) for the FGD wild-type and mutant enzymes. A Spectrosil Quartz submicro cell (160 μL nominal volume) from Starna Cells was used to monitor the binding assays at 22 °C on a Horiba FluoroMax Spectrofluorometer, with excitationand emission slit widths set at 4 and 8 nm, respectively. All binding assays were performed in 50 mM Tris-HCl (pH 7.0). To obtain the Kd of F420, increasing concentrations of F420 in either 1 or 2 μL aliquots were titrated into a solution containing 350 nM FGD, to final F420 concentrations of 0.05−8.8 μM. The samples were excited at 290 nm, and the emission scan was performed from 310 to 500 nm. The Kd for G6P was also determined under similar conditions by titrating 1 or 2 μLaliquots of G6P into a solution containing 350 nM FGD, to final G6P concentrations of 2−371 μM. Each experiment was conducted in duplicate. |
| Affinity data for this assay | |
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