Table 2.

Table showing the inputs for fitting Eq. (11) in GraphPad Prism software. Columns include parameter definitions, constraints on the fit parameters, initial value predictions for each parameter, and the results of the fit. FA = fluorescence anisotropy. Numerical values shown in the table are unitless unless otherwise noted as micromolar concentrations.

Parameter	Description	Constraint	Initial value	Fit result
Kd	The dissociation constant	Must be greater than 0	0.8 (μM) (EC50 from Eq. (3))	1.5 ± 0.33 (μM)
S	Total ligand concentration	=0.010 (μM)	Fixed	0.010 (μM)
rb	FA signal for maximally bound complex	Must be greater than 0	“YMAX” (maximum FA value)	164 ± 4
rf	FA signal for 100% free ligand	Must be greater than 0	“YMIN” (minimum FA value)	60 ± 6
R	Fluorescence intensity correction factor	=1.8	Fixed (R from Eq. (5))	1.8