Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

Abstract In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C44), 83% carbamylated (C83) and 85% acetylated (A85) were made by treating the HSA solution with a different molar excess of O-methylisourea and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl HR 200 column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C83 and A85 HSAs while C44 HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 nm to a different extent. Whereas the values of the Stern-Volmer constant (KSV) and bimolecular quenching rate constant (kq) suggested, NA-HSA complex formation, binding constant (Ka) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the Ka value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.