Comparison of graphical procedures for estimating the intrinsic molar fluorescence of protein-bound drugs for drug-binding studies. A reevaluation of existing plots and introduction of two inverse hyperbolic plots.

A theoretical evaluation of existing and new graphical procedures for estimating the intrinsic molar fluorescence (psi B) of a protein-bound drug has been undertaken. The results do not concord with a recent proposition that psi B should be obtained by direct reading from a graph of emitted fluorescence intensity (I) against the logarithm of the binding protein concentration ([P]) rather than by extrapolation of a double reciprocal plot. The calculated errors in estimates of psi B obtained by direct reading or by extrapolation of standard plots and of three new inverse hyperbolic plots showed that, independently of binding affinity: 1. Direct reading from the logarithmic plot gave the least accurate estimates. 2. The single reciprocal plot gave more accurate estimates than the double reciprocal plot providing the (constant) drug concentration was similar to or greater than its dissociation constant in the binding system. At lower drug concentrations the double reciprocal plot gave more accurate estimates. 3. Extrapolation of an inverse hyperbolic sine plot (sinh-1 (1/I) against 1/[P]) did not give more accurate estimates than the standard reciprocal plots. 4. If the drug concentration was close to its dissociation constant the most accurate estimates were obtained with an inverse hyperbolic cosine plot of cosh-1 (I + 1) against 1/[P]. For a low affinity binding system in which non-specific binding is significant an inverse hyperbolic sine plot of 1/sinh-1I against 1/[P] gave the most accurate estimates at low drug concentrations. An experimental and theoretical procedure for optimizing the estimation of psi B is proposed on this basis.