Determination of binding dissociation constants and stoichiometric coefficients of cofactors and WY 14,643 to hAR using fluorescence titration data

The methodology described by van de Weert 2010 was followed in the binding constant determination^27,28. The fractional saturation (α) of protein by cofactors (NADP⁺/NADPH) and by inhibitor (WY 14,643) binding to protein can be expressed in the form of fluorescence as follows;

where F⁰ is the fluorescence in absence of quenchers (cofactors and inhibitor) denoted by L. F is the fluorescence at a given quencher concentration and F^∞ is the fluorescence from a protein “saturated” of quencher. Assuming that quenchers bind to protein with molar stoichiometry of 1:n as shown in the equation (2);

where n represents the order (stoichiometry) of kinetic reaction, k₊₁ and k₋₁ represent binding and dissociation rate constants respectively. The equilibrium dissociation constant can be expressed as below;

At equilibrium;

The equilibrium dissociation constant can be expressed as below;

This equation can be re-arranged to,

when quencher concentration (L) ≈ Initial concentration (L⁰) then equation (8) can be expressed as follows;

The binding dissociation constants and stoichiometry for the binding of quenchers (cofactors and inhibitor) to protein were calculated by non-linear fitting to the experimental fluorescence data and results are shown in Table 1.