The binding of irbesartan to bovine hemoglobin (BHb) has been investigated for the first time by using UV-Vis absorption, fluorescence, circular dichroism (CD), and molecular docking. The binding site number
A full understanding of the modes of drugs’ action requires the study on the interactions of all possible chemical and biological targets, including amino acids, hormones, peptides, and proteins. Studies based on drugs-protein interactions improve the armory of molecular tools for detecting and manipulating the biological roles of biomacromolecules. Such studies also crucially determine the bioavailability and toxicology of some injected drug. Hemoglobin (Hb) is an important functional protein for reversible oxygen carrying and storage in animals. It also transports carbon dioxide to regulate the pH of blood. Hb contains four globin chains, the two of them are
Irbesartan (Figure
The chemical structure of irbesartan.
Study on the interaction of drugs to proteins can elucidate the properties of drug-protein complex. For a better understanding, the study of interaction between irbesartan and Hb is much needed. The mode of interaction, binding constant, and binding sites are significant in our research. To characterize the interaction at the molecular level, optical techniques are great tools because of their high sensitivity, rapidity, and ease of implementation. The flexible protein-ligand docking plays an increasingly important role in the identification of potential lead compounds in the drug discovery process. It can dock flexible ligands into an ensemble of protein structures which represents the flexibility, point mutations, or alternative models of a protein [
In this paper, the fluorescence quenching technique and UV-Vis absorption spectra were applied to study the interactions of irbesartan and Bovine hemoglobin (BHb). Furthermore, far-ultraviolet circular dichroism (CD) spectroscopy was used for demonstrating the changes of protein secondary structure. Finally, in order to obtain a clearer insight into the residues involved in the interaction, the irbesartan binding mode to BHb was studied using automated flexible docking approach. Experimental observations and theoretical data could be helpful to understand transports of irbesartan in the blood.
Bovine hemoglobin (BHb) was purchased from Sigma Chemical Company and used without further purification. Irbesartan was obtained from Shanghai Institute of Materia Medica Chinese Academy of Sciences. BHb solutions (1 × 10−5 M and 5 × 10−6 M) were prepared in 0.05 M Tris-HCl buffer solution with PH 7.4 containing 0.1 M NaCl to maintain the ionic strength of the solution. The irbesartan solution (1 × 10−4 M) was prepared by dissolving irbesartan in absolute methanol solution. Absolute methanol was purchased from TEDIA Company in America. All other reagents and solvents were of analytical reagent grade. All aqueous solutions were prepared using newly double distilled water.
UV-Vis spectra of samples were recorded on a Cary 50 spectrophotometer equipped with 1.0 cm quartz cell at 298 K, using Cary Winuv software.
A Cary Eclipse spectrofluorimeter (Varian, Australia) equipped with 1.0 cm quartz cells was utilized to measure the fluorescence emission spectra, using 5.0 nm×10.0 nm slit widths. Different ratios of irbesartan and BHb solutions were prepared before the experiments. They were 0, 0.1, 0.2, 0.4, 0.8, and 1.2, respectively. The emission spectra were recorded in the range of 300–400 nm using the excitation wavelength of 280 nm.
The CD spectrums were obtained on a Jasco J-810 spectropolarimeter (Jasco Co., Ltd., Tokyo, Japan). The samples were transferred to a quartz cell with a 1 cm light-path length. Molecular ellipticities were measured in the range of 200–225 nm. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before using and kept in nitrogen atmosphere throughout the experiment.
The crystal structure of BHb used for molecular docking was obtained from the structure having Protein Data Bank (PDB) identifier 1G09. In order to optimize hydrogen positions, remove interatomic bumps, and correct the covalent geometry, the structure of BHb was energy-minimized with the CHARMm force filed. Waters and all other HETATM molecules were removed from the BHb PDB file. Polar hydrogen atoms and Gasteiger charges were added to prepare the BHb molecule for docking. Protein-ligand docking was carried out with the flexible docking tool in the Discovery Studio 2.1 (DS 2.1) software of Accelrys company in America. The flexible of the residues which surround the active site of BHb and the ligand were all considered in the docking procedure. In this work, the selection of flexible residues for the induced fit is based on the active site of the BHb.
UV-Vis absorption measurement is a very simple and applicable method to explore the structural change which may be caused by the complex formation. The UV-Vis absorption spectra of BHb with and without irbesartan were showed in Figure
UV-Vis absorption spectrum of (a) BHb and (b) irbesartan. BHb = 0.4 system.
For macromolecules, the fluorescence measurements could provide some information of the binding of small molecule substance to protein on the molecular level, such as the binding mechanism and binding constants, In order to ascertain the value of the association constant and the binding sites in the interaction between irbesartan and BHb, fluorescence spectroscopy is much needed. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule, such as excited-state reaction, molecules rearrange, energy transfer, ground state complex formation, and collision quenching. The fluorescence quenching behavior is known to occur mainly through a dynamic process or a static process. Collisional or dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to the formation of the complex between quencher and fluorophore.
Figure
Emission fluorescence spectra of BHb with a concentration ratio of irbesartan and BHB: (a) 0, (b) 0.1, (c) 0.2, (d) 0.4, (e) 0.8, and (f) 1.2.
Figure
Stern-Volmer plots for the interaction of irbesartan and BHb.
The fluorescence quenching came from the formation of complex between irbesartan and BHb, then the equilibrium between free and bound molecule could be given by following equation [
Plots of log [
In order to get a better understanding in physicochemical properties of irbesartan governing its spectral behavior and to draw relevant conclusions on the irbesartan-BHb binding mechanism, CD spectroscopic measurements were performed on BHb and the irbesartan-BHb complex. If the change of protein structure included the transforming of protein secondary structure in the drug-protein complex, it can be reflected in the CD spectra. In Figure
CD spectra of the BHb-irbesartan system. (a) 5 × 10-6 M BHb; (b) irbesartan:BHb = 0.4; pH 7.4.
There are four chains in the BHb molecule, named A, B, C, and D. For the docking experiment, the cavities in the BHb were defined as the active sites of BHb. The number of 15 active sites has been found in the BHb molecule according to the docking procedure. To establish which binding site of BHb that irbesartan is located in, the complementary applications of molecule docking have been employed by computer methods to improve the understanding of the interaction of irbesartan and BHb. The molecule docking was investigated through the flexible docking protocol in the DS 2.1 software.
Flexible docking means that the side chains can rotate continuously about single bonds during the docking simulation. The active-site atoms of the receptor were defined as those atoms within a radius of 9.0 Å from the ligand co crystallized with that particular receptor. Here, the conformational space of the ligand irbesartan is explored within the defined active-site of the BHb. The nine ligand poses were generated by the flexible docking protocol, and all the irbesartan structures were scored by means of the libdock scoring function and scored in increasing orders of their scores. The higher the libdock score, the better the energy ranked [
The interaction between irbesartan and BHb, (a) an overview of the irbesartan molecular (stick molecular) located in BHb; (b) the binding mode between irbesartan and the defined flexible residues in the BHb.
Figure
In Figure
In the present work, the binding of irbesartan and BHb under physiological conditions has been studied using molecular docking and several optical techniques. Experimental results showed that the binding between irbesartan and BHb induced a conformational change of BHb, which was further proved by UV-Vis and CD spectrum. The results of fluorescence quenching measurements and molecular docking study suggested that irbesartan could bind to BHb through the hydrophobic interaction and hydrogen bond with high affinity. The binding site is located in the site 2 pocket of BHb according to the molecular docking study. The CD spectroscopic showed that the secondary structure of BHb changed after irbesartan bound to BHb. The number of binding site and the binding constant between the drug and BHb was calculated based on the fluorescence experiment results. The binding study of irbesartan with BHb is of great importance in pharmacy, pharmacology, and biochemistry. This experiment can supply important information to clinical research and provide the theoretical basis for drug designing.