The Stern-Volmer equation for fluorescence quenching is Fo/F = 1 + Ksv[Q].

I'm not sure this is the right equation to use for measuring quenching of intrinsic fluorescence of BSA by drug binding. The Stern-Volmer equation applies to quenchers in free solution, whereas quenching by drug binding is saturable due to the fact that the number of drug binding sites is limited by the amount of BSA.

I would plot fluorescence intensity versus drug concentration, then use non-linear regression to fit the curve to a binding model such as F = Fo - A[L]/(Kd + [L]), where [L] is drug concentration, Kd is the dissociation constant of the drug, and Fo-A is the fluorescence at [L]>>Kd (i.e. once the binding site is saturated). A more complex model may be needed because BSA has multiple drug binding sites.

You should also measure the fluorescence of the same titration but without BSA in order to measure the fluorescence of the drug and subtract it from the titration with BSA. You should also measure the inner filter effect of the drug, which is the absorbance of the excitation and emission light in order to correct for that, if necessary. You can do that using N-acetyltryptophanamide as the fluorophore instead of BSA, under the assumption that the drug does not bind to it.

Dear Adam,

Thank you very much for your detailed answer. I would like to point out that the equation I used is Lehrer's modification of Stern-Volmer (Lehrer 1971; Lehrer et al 1978), which can be found in many publications (an example paper attached).

I've used non-linear regression of Scatchard's equation and calculated Kd (dissociation constant) and n (number of binding sites). I've also calculated KSV from the Stern-Volmer equation you've stated. I thought I would also calculate the fa value from modified Stern-Volmer but as I've mentioned, for some ligands it was greater than one.

OK, that is new to me. Make sure you are not getting interference from the compound of either type: compound fluorescence or fluorescence quenching, as I mentioned previously.