Characterization of the drug-binding site "Sudlow I" of human serum albumin using anthranilic acid probe to reveal the role of Trp-214 in the binding mechanism
Author
Al-Busaidiyah, Badriya Yahya Saleh
English abstract
Anthranilic acid (AA) was used in this project to probe the drug-binding site Sudlow I in subdomain IIA of human serum albumin (HSA) using steady-state and time-resolved spectroscopic measurements. The spectroscopy of AA was first characterized in different solvents of varying polarity and hydrogen bonding capability. Analysis of the absorption and fluorescence spectra reveals that AA exists as an anion in water only. The Stokes shift of AA in different solvents was found to be linearly correlated with the normalized molar transition energy of solvent polarity (EM). The longest lifetime of AA was measured in water and is attributed to solvation of the polar groups of the molecule. Excimer formation containing two AA molecules stabilized by intermolecular hydrogen bonds was observed at a high concentration of AA in water.
Characterization of the drug-binding site of HSA was performed by measuring the change in fluorescence of both AA and of HSA itself upon protein ligand recognition. A large quenching in the fluorescence intensity of HSA and a reduction in its fluorescence lifetime were observed upon the addition of AA. The results indicate that AA interacts with the tryptophan residue (Trp-214) in subdomain IIA of HSA. The reduction in the fluorescence of Trp-214 is due to energy transfer from this residue to AA. The calculated distance between Trp-214 and AA using Förster's theory of energy transfer and the estimated quenching rate constant using a Stern-Volmer plot both point to a major contribution from static quenching to the quenching mechanism in the AA:HSA complex. Binding of AA in HSA was also
udied using a binding isotherm and AA was found to selectively bind in subdomain IIA only up to a concentration of 10 times that of HSA. Selective interaction between AA and Trp-214 results in partial unmasking of fluorescence from tyrosine. The measured steady-state and time-resolved anisotropy of AA in solution and in the HSA protein indicates that the AA molecule is caged inside the binding site of HSA and its free rotation is restricted. A slight decrease in intensity and a blue shift in the AA fluorescence peak were observed in the presence of HSA. The results are explained to be due to a direct interaction between AA and Trp-214 in the excited state.
Chemical unfolding of HSA in guanidine hydrochloride (GdnHCl) was found to start at GdnHCl concentration at 2.0 M and is complete at 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to fluorescence from Trp-214 in a polar environment and the other peak was assigned to fluorescence from tyrosine. In the presence of AA, both fluorescence peaks were slightly quenched which strongly support the assignment of the tyrosine peak to be due mainly to the Tyr-263 residue in subdomain LA. Finally, dilution refolding of denatured HSA was examined. The results indicate that the refolding mechanism is reversible without the need for any refolding agent. The presence of AA during the processes of unfolding and refolding causes only slight quenching in the HSA fluorescence. The results indicate that the small size of the AA molecule is hardly causing any deformation to the protein structure which makes this molecule a suitable probe to study binding sites in proteins and in other supramolecular systems.
