Analysis of the binding of fluorescent ligands to soluble proteins: Use of simultaneous non-linear least squares regression to obtain estimates of binding parameters

The binding of three fluorescent ligands (warfarin, dansylsarcosine and 1-anilino-8-naphthalene sulphonate) to human albumin was analysed using simultaneous non-linear least squares regression analysis. Both mock and actual fluorescence data were examined and the results indicated that reliable estimates of the binding parameters as well as the molar fluorescence of bound ligand could be obtained. The advantage of this method of analysis is that it makes full use of all the experimental data and it eliminates the need for the graphical procedures usually employed to estimate the molar fluorescence of bound ligand and its binding constants. This type of analysis can be extended to other systems where some physical property of the bound ligand varies with increasing protein concentration.