2.1.

Materials

Rifampicin was purchased from Sigma-Aldrich (Steinheim, Germany). Phosphate-buffered saline (PBS) solution, acetate buffer, BSA, ethanolamine-HCl, N-hydroxysuccinimide (NHS), and N-ethyl-N-(3-diethylaminopropyl) carbodiimide (EDC) were purchased from Merck (Darmstadt, Germany). Carboxymethyl dextran (CMD) low-density-modified gold surfaces chip was prepared from Bionavis Company (Tampere-region, Finland).

2.2.

Apparatus

All kinetic parameters were recorded with dual-flow channels SPR device (MP-SPR Navi 210A, BioNavis Ltd., Tampere-region, Finland).

2.3.

Bovine Serum Albumin Immobilization

After putting a CMD sensor chip on SPR instrument, the whole device is rinsed with acetate buffer. As soon as reaching steady baseline in sensorgram, the chip surface cleaned with NaCl (2 M) and NaOH (0.1 M) by injecting them to both channels for 3 min. To activate the chip surface EDC: NHS 1:1 (NHS 0.05 M + EDC 0.2 M) solution injected to the sensor chip for 7 min. For immobilization of BSA, the BSA solution injected ($0.3\mathrm{mg}/\mathrm{ml}$) to channel 1, for 10 min and acetate buffer injected (running buffer) to channel 2 (reference channel). Finally, ethanolamine-HCl (1 M, pH 8.5) was injected for blocking the remaining activated surface groups. All of the injection processes were performed with flow rate of $30\mu \mathrm{l}/\min$.

2.4.

Kinetic Parameters of Bovine Serum Albumin Interaction with Rifampicin

Rifampicin solution in PBS buffer at pH 7.4 was injected using a flow rate of $30\mu \mathrm{l}/\min$ for 3 min at various concentrations (1 to $128\mu \mathrm{M}$). Both flow cells were used for sample injection and due to BSA immobilization at one flow cells, the flow cell without any BSA was used as reference. Rapid dissociation of rifampicin from BSA surface omits the need for regeneration process.

To estimate the affinity and kinetic of the BSA interaction with rifampicin, Trace Drawer^™ for SPR Navi^™ was used. Before calculations in Trace Drawer, data are extracted with SPR Navi^™ Data viewer software.

2.5.

Thermodynamic Analysis

The effect of temperature on binding of rifampicin to BSA was studied to estimate the thermodynamic parameters of rifampicin–BSA complex formation. Therefore, SPR measurements were investigated at four different temperatures (298, 303, 308, and 313 K).

2.6.

Molecular Docking Simulation

The docking study of BSA with rifampicin was performed by the open-source windows version of Auto Dock Vina 1.1.2 software. Auto Dock Vina not only improves the accuracy of molecular docking between the ligand and receptor but also increases the speed of dock running.¹⁵ For study of the molecular interaction of BSA with rifampicin by in silico simulation, the x-ray structure of BSA (PDB ID code4F5S, 2.47 Å resolutions) was selected from the Research Collaboratory for Structural Bioinformatics protein data bank. The rifampicin molecular structure was prepared by drawing the two-dimensional chemical structure by Chem Draw Ultra 10.0 software and then for molecular energy optimization, the HyperChem 8.0.8 software was selected. Molecular structure optimization of rifampicin was done in two steps: at first the MM+ molecular mechanics was performed and after completed this step the AM1 semiempirical procedures was executed. To minimize rifampicin structure, the Fletcher–Reeves algorithm was selected in both optimization processes.¹⁶ For the docking study, the extracrystallographic structure, such as water molecules, was removed from the BSA structure. The BSA structure and compound were changed by adding the polar hydrogens. Also, the Kollman charges as well as Gasteiger charges were added to the receptor and ligand, respectively. After attaining the pdbqt files of BSA and rifampicin, drug was inputted in determined six different binding sites of BSA. The grid box dimension size at grid points in $x\times y\times z$ directions were set to 27 Å with a grid spacing of 1 Å in Vina docking. The energy range and exhaustiveness value were set to $4\mathrm{kcal}/\mathrm{mol}$ and 40, respectively.

3.1.

Bovine Serum Albumin Immobilization

Figure 2 shows the schematic illustration of BSA immobilization on the Au chip surface.

Fig. 2

Schematic illustration of BSA immobilization by amine coupling: (a) carboxymethyl dextran sensor chip, (b) activation of COOH in CMD via EDC/NHS before BSA immobilization, (c) immobilization of the BSA through covalent amide bond formation, and (d) deactivation of the remaining activated surface groups by blocking reagents ethanolamine, which is the most commonly used agent.

The initial curve of the aforementioned process before and after BSA immobilization was shown in Fig. 3. The blue curve shows channel 1, in which BSA has been immobilized and showed angle shift around 0.98 deg. The resonance curves shift to higher incident angles and become wider due to new layer formation on Au sensor slide and increase in the thickness of the adsorbed layer on the surface. From the aforementioned results, it can be concluded that the BSA attachment was performed properly on the Au sensor slide through covalent amide bond formation.¹⁷

Fig. 3

Initial SPR curve (a) before and (b) after BSA immobilization.

3.2.

Kinetics Binding of Bovine Serum Albumin and Rifamipcin

The interaction between the analyte ($A$) and the immobilized BSA ($B$) can be demonstrated through simple Langmuirian mode of binding for complex formation ($AB$). For reversible associations and dissociations in a chemical equilibrium, it can be defined by the following equation:¹⁷

Fig. 4

Sensorgram of rifampicin interaction with immobilized BSA at various concentration 1 to $128\mu \mathrm{M}$ at different temperatures (a) 298, (b) 303, (c) 308, and (d) 313 K.

Table 1 shows values of kinetic parameters at four temperatures. According to the Table 1, $k_{\mathrm{a}}$ and $k_{\mathrm{d}}$ of BSA interaction with rifampicin decreased regularly upon rising temperature. It shows that high temperature led in the reaction rate decrease. The mean $K_D$ for rifampicin binding increased almost 2.30 from 15 to $34.6\mu \mathrm{M}$ over this temperature range. The temperature dependence of the $K_D$ was due to an increase in the $k_{\mathrm{d}}$ value rather than the $k_{\mathrm{a}}$ value. About 15-fold increase in the $k_{\mathrm{d}}$ value was observed when temperature increased from 298 to 313 K. However, there was only sixfold increase in the $k_{\mathrm{a}}$ value when temperature rose to 313 K. As it is clear in Table 1, $K_D$ of BSA interaction with rifampicin increased upon rising temperature, which showed that affinity between two materials decreases upon rising temperatures.

Table 1

Association rate constant (ka), dissociation rate constant (kd), and equilibrium constants (kD) values of rifampicin interaction with BSA.

3.3.

Thermodynamic Parameters Analysis of Binding Bovine Serum Albumin with Rifampicin

To investigate the effect of temperature and thermodynamic features, the reaction between BSA and rifampicin was carried out at different temperatures. The type of interaction between BSA protein and rifampicin may consist of the following forces: hydrogen bond, Van der Waals, electrostatic, and hydrophobic forces. The value of thermodynamic parameters, such as enthalpy change ($\mathrm{\Delta }H$) and entropy change ($\mathrm{\Delta }S$), is used for the estimation of interaction types. The positive value of $\mathrm{\Delta }H$ and $\mathrm{\Delta }S$ shows that the hydrophobic forces play major roles in the binding of two molecules; the negative values represent Van der Waals interactions and hydrogen bonds occurring and finally, the negative value of $\mathrm{\Delta }H$ and positive amount of $\mathrm{\Delta }S$ is indicative of electrostatic interaction happening. These parameters can be calculated through the van’t Hoff Eq. (1) by the plot of ln $K_D$ versus $1/T$¹⁹

BSA binding study was performed at four different temperatures (298, 303, 308, and 313 K). Figure 5 shows the plot of ln $K_D$ versus $1/T$.

Fig. 5

Van’t Hoff plot for the interaction of BSA and rifampicin.

The total energy released by the reaction is calculated with the Gibbs–Helmholtz

Table 2

Thermodynamic parameters of BSA binding with rifampicin at four different temperatures (298, 303, 308, and 313 K).

3.4.

Molecular Docking Study

Twenty mode runs were defined in our docking procedure. After molecular docking process, the best conformation of each rifampicin–BSA complexes relative to the lowest free binding energy ($\mathrm{\Delta }G$) according to the Vina cluster was selected and summarized in Table 3 and the lowest free binding energy conformer of docking for each binding site has been shown in Fig. 6. The lowest free binding energy ($\mathrm{\Delta }G$) for the sites $A$ and $B$ of albumin molecule was found and presented in Table 3. The subdomain IIB and IIA of albumin molecule has some amino acids with functional groups that can make hydrogen binding as well (Fig. 7). Our docking analysis revealed some H-bondings between rifampicin and BSA (Table 4). Rifampicin has also several functional groups, such as hydroxyl with hydrogen binding capability. Therefore, the possibility of some hydrogen bindings between rifampicin and BSA cannot be totally excluded, although based on the results obtained from thermodynamic studies, this is the hydrophobic forces that act as the major driven forces in the interaction of rifampicin with the protein. Therefore, it seems that rifampicin interacts with BSA through hydrophobic interactions as the major driven forces.^1,21

Table 3

The lowest binding free energy values (ΔG) calculated by Auto Dock Vina from rifampicin docking with six different binding sites of BSA.

Fig. 6

The best docked conformation poses of rifampicin in six various binding sites of BSA.

Fig. 7

H-bonding between rifampicin and different amino acids in two binding subdomains of BSA. Green spheres are H-bonding and wireframe spheres are close contact between rifampicin and residues.

Table 4

Amino acids that contacted with rifampicin from the analyzing of all runs of each docking procedure and the number and type of amino acids that join together in formation of H-bond in selected binding site of BSA (IIA and IIB). The number of phrases indicated the repeating of H-bonding with dependent amino acid.