Effect of ligands with different affinity on albumin fibril formation

The effect of binding of several ligands to bovine serum albumin on the kinetics of fibril formation at denaturing conditions is studied. The considered ligands are clinical drugs with different binding constants to albumin: relatively strong binders (naproxen, ibuprofen, warfarin with 10⁵ to 10⁷ binding constant values) and weak binders (isoniazid, ranitidine with 10³ to 10⁴ binding constant values). The data of thioflavin fluorescence binding assay, Congo red binding assay, and circular dichroism spectroscopy indicate ligand concentration-dependent suppression of fibril formation in the presence of strong binders and no effects in the presence of weak binders. Analysis of kinetic curves shows no induction lag associated with fibril nucleation and the first-order kinetics of fibril formation with respect to albumin concentration for all the studied systems. Using DSC method, the fractions of unfolded albumin at incubation temperature were determined for each albumin-ligand system and ligand concentration. Their magnitudes ranging from 0 to 1 correlate with the initial rates of fibril formation and with equilibrium concentrations of fibrils formed in the system after incubation for at least 120 min. The results indicate that fibrils are formed from partially or completely denatured albumin form with the rate proportional to the fraction of this form. Strong albumin binders act as thermodynamic inhibitors of fibrillation shifting the unfolding equilibrium to the side of the native ligand-bound protein.