Exploring ligand-protein interaction: A laboratory exercise on herbicide binding to plasma transport protein

A laboratory exercise on the interaction between the herbicide pendimethalin (PM) and goat serum albumin (GSA), a carrier protein present in mammalian blood circulation, is described. Fluorescence spectroscopy was used to study the binding reaction between PM and GSA. Titration of a constant amount of the protein (GSA) with increasing ligand (PM) concentrations produced a consecutive decrease in the protein's fluorescence. Treatment of the fluorescence quenching data according to the Stern–Volmer equation yielded the values of the Stern–Volmer constant (K sv ) and bimolecular quenching rate constant (k q ), whereas values of the binding constant (K a ) and number of binding sites (n) were obtained from the double logarithmic plot. This experiment provides an exciting opportunity for undergraduate students to independently perform ligand binding studies with a protein, in addition to providing the means for the determination of their binding parameters. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 156–160, 2019. © 2019 International Union of Biochemistry and Molecular Biolog

Biophysical and computational approaches to unravel the molecular interaction mechanism of bromodeoxyuridine, a proliferative marker with human serum albumin

Abstract: The interaction of a nucleoside analogue bromodeoxyuridine (BrdU) with human serum albumin (HSA) was studied to investigate the binding phenomenon and analyse the protein conformation upon BrdU binding. Multiple spectroscopic techniques, viz. intrinsic and 3-D fluorescence, UV–Vis absorption, and circular dichroism (CD) spectroscopy along with molecular docking were used. Decrease in the Stern–Volmer constant (Ksv) as well as the association constant (Ka) with increase in temperature suggested BrdU–HSA complex formation. Intermediate binding affinity between BrdU and HSA was evident from the Ka values (2.49–3.97 × 104 mol–1 dm3), while BrdU–HSA complex formation was driven by hydrophobic and van der Waals interactions along with hydrogen bonds, as revealed by thermodynamic data (ΔS = + 28.48 J mol−1 K−1; ΔH = − 17.16 kJ mol−1). Minor changes occur in both secondary and tertiary structures as well as in the fluorophores’ microenvironment of HSA, as recognized from the CD spectral results in the far-UV and the near-UV regions and 3-D fluorescence spectra, respectively. Use of site markers (warfarin and indomethacin for site I; diazepam for site II) as well as docking results suggested BrdU binding to both site I (more preferred) and site II, located in subdomains IIA and IIIA, respectively, of HSA. Graphic abstract: [Figure not available: see fulltext.]. © 2019, Springer-Verlag GmbH Austria, part of Springer Nature