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

• Published in: Journal of the Indian Chemical Society Vol. 98; no. 3; p. 100031
• Main Authors: Tan, Cheau Yuaan, Lim, Chun Shen, Liew, Siew Mun, Abd Halim, Adyani Azizah, Tayyab, Saad
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-03-2021
• Subjects: Chemical modification; Drug-protein interaction; Human serum albumin; Nalidixic acid; Human serum albumin; Chemical modification; Drug-protein interaction; Nalidixic acid
• ISSN: 0019-4522
• DOI: 10.1016/j.jics.2021.100031

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 potassium cyanate 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 S-200 HR 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.
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•Similar molecular expansion in 83% carbamylated and 85% acetylated HSA derivatives.•Retention of the native protein conformation in 44% carbamylated HSA.•Nalidixic acid-HSA complex formation with moderate binding affinity.•Importance of lysine residues of HSA in nalidixic acid binding.