Quantitative effect of shape parameters a and b on practical identifiability of three-site binding parameters. (A–E, left) Three-site total binding curves computed using the indicated values of shape parameters a and b. Location parameter p₁ was equal to (in M⁻¹) 10¹⁰ (A, solid curve), 10⁷ (A, dashed curve), 10¹⁰ (B, solid curve), 10⁷ (B, dashed curve), 10¹⁰ (C, solid curve), 10⁷ (C, dashed curve), 10⁹ (D, solid curve), 10⁶ (D, dashed curve), 10⁷ (E, solid curve), and 10⁴ (E, dashed curve). (A–E, middle) Location parameter p₁ fit-error plots for binding curves in A–E (left). (A–E, right) Fit-error plots for shape parameter a (red) and shape parameter b (blue) for binding curves in A–E (left). Dashed horizontal lines in A–E (middle and left) indicate 5% error threshold. Binding parameters are denoted “PI” if the asymptotic value of the rms fit error is ≥5% and “NOT PI” if <5%.