Mechanistic and kinetic aspects of Natamycin interaction with serum albumin using spectroscopic and molecular docking methods

2.1 Material

N-ethyl-N-(3-dimethyl aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), phosphate-buffered saline (PBS) solution, and ethanolamine-HCl was bought from Merck (Darmstadt, Germany). Acetate buffer, NT, and BSA was bought from Sigma-Aldrich. Carboxymethyl dextran (CMD) low-density-modified gold surface chips were bought from BioNavis Company (Tampere region, Finland). Other materials utilized were of the highest quality.

2.2 Stock preparation

Phosphate buffer was prepared by dissolving a tablet of saline phosphate buffer in deionized water. The stock solution of BSA (1.4 $\times$ 10⁻⁵ M, according to its molecular weight of 66,000D) was dissolved in a 0.01 M phosphate buffer solution (pH 7.40). NT stock solution at a concentration of 2 mM was prepared by dissolving 1.4 mg NT in PBS buffer solution. All ready solutions were stored in the refrigerator at 4 °C.

2.3 Fluorescence experiments

Fluorescence spectroscopy evaluations were performed at five distinct temperatures with a Jasco FP-750 fluorescence spectrometer (Jasco International Co, Kyoto, Japan) equipped with a thermostat. The measurements were done in a 10 mm quartz cell. To observe the fluorescence of Trp residues, a wavelength of 290 nm was used for excitation. The slit width for excitation and emission was 5.0 nm. The scanning speed of the device was set to 8000 nm/min. 1 mM BSA solution was titrated with concentrations of 1⁻⁶ × 10⁻⁵ and its fluorescence spectrum was recorded at a wavelength of 300–450 nm. All measurements were performed with PBS buffer at 288, 293, 398, 303, and 310 K. To eliminate the inner filter effects of protein and ligands, absorbance measurements were performed at the excitation and emission wavelengths of the fluorescence measurements. The fluorescence intensity was corrected using Eq. (1):

2.4 UV–Vis spectrophotometry

To record Absorption spectra of BSA without and with different concentrations of NT at a temperature of 25 °C, a UV–Vis spectrometer model T70 + manufactured by PG Instrument Company (Leicestershire, UK) was used. Titration of 1 mM BSA solution with distinct concentrations of NT (0–70 μM) was performed in a 10 mm quartz cuvette. Finally, the solution was thoroughly blended and incubated for up to 3 min to let NT interact with BSA.

2.5 SPR measurements

To investigate the interaction of BSA with NT and calculate the kinetic parameters, an SPR device (MP-SPR navi210 A, Bionavis Ltd, Tampere, Finland) with a dual-stream channel (using Kerchman's prism configuration) and a continuous peristaltic pump was used (Sharifi et al., 2017).

2.6 Immobilization of BSA on CMD sensor chip

The CMD sensor chip was settled within the SPR instrument. Acetate buffer (10 mM) with pH 4.5 was utilized to wash the entire machine. After 15 min, the sensor diagram reached the baseline, then NaCl (2 M) and NaOH with concentration (0.1 M) were used to clear the SPR channels for 3 min at a speed of 30 μl/min. In the next step to activate the carboxyl groups on the CMD sensor chip, a stream of solution (NHS 0.05 M + EDC 0.2 M) EDC/NHS in equal ratios of 1:1 was injected into the SPR instrument at a speed of 30 microliters per min for 7 min. Then, to stabilize BSA on the CMD sensor chip, a solution of BSA at a concentration of 1 mg/ml was infused into channel 1 for 7 min and acetate buffer was infused simultaneously into channel 2. Finally, a stream of Ethanolamine-HCl solution (1 M, pH 8.5) was injected at an amount of 30 μl/min to inactivate and block the active carboxyl groups on the surface of the CMD chip (Sharifi et al., 2017; Fathi et al., 2019).

2.7 Kinetic parameters analysis of BSA interaction with NT

To calculate the kinetic parameters including association constant (K_a), dissociation constant (K_d), and equilibrium constants (K_D), which are calculated via $K_D=k_d/k_a$ , different concentrations of NT were prepared in PBS buffer with pH 7.4 and stream of the prepared solution at a speed of 30 μl/min for 2 min at 293, 298, 303, and 310 K was injected into both flow cells. Since BSA was immobilized in only one channel, both stream cells were utilized for sample injection and the stream cell without BSA was utilized as a reference. The need for the regeneration process is eliminated due to the rapid separation of NT from the BSA level. By measuring the SPR signal, the amount of binding of NT to BSA was determined. Drawer^TM for SPR Navi^TM was used to determine the kinetic parameters upon BSA interaction with NT (Maleki et al., 2020).

2.8 Study of molecular docking

To understand the possible interaction of NT with BSA as a potential target biomolecule, a molecular docking study was conducted via the AutoDock 4.2 package, which is validated by The Scripps Research Institute, La Jolla, CA, USA. The 3D molecular building of NT was achieved from the PubChem website (PubChem CID:5284447) (https://pubchem.ncbi.nlm.nih.gov/), and following that, its structure was optimized for our calculation through Gaussian 03 software (B3LYP method). Next, the BSA crystal structure (PDB code: 3 V03, 2.7 Å) was downloaded from Protein Data Bank Website (https://www.rcsb.org/). AutoDock Tools-1.5.6 program was used to apply Kollman/Gasteiger charges to the BSA structure (subunit A), and also eliminate all water molecules. To find the binding domain on BSA, a blind docking calculation was performed at a lace box of 126 × 126 × 126 Å (spacing parameter of 0.523 Å), and then the lowest binding score was considered for further analysis. To focus on the binding site, another docking study was accomplished at a grid box of 70 × 70 × 70 Å and a spacing parameter of 0.380 Å. The Lamarckian genetic algorithm was applied in all calculation runs. The results were displayed and analyzed via UCSF Chimera software (version 1.14).

3.1 Study of BSA fluorescence quenching

The fluorescence quenching analysis was used for the investigation of NT interaction with BSA. It is a useful procedure for the study of protein interaction with various molecules and the evaluation of structural changes induced in the protein on the molecular level (Wani et al., 2018). Among the different types of molecular interactions, we can mention excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and quenching collisions that lead to quenching (Lakowicz, 2006). In general, the innate fluorescence of BSA is inferred from Trp, Tyr, and Phe. However, among them, the contribution of tryptophan is a maximum (Wu et al., 2019; Javaheri-Ghezeldizaj et al., 2021). Innate fluorescence of BSA is regularly utilized as a test to find the affinity of BSA to various food additives due to the Trp residues presence on the surface of BSA (Trp 134) and the interior of the hydrophobic pocket (Trp 213).

The fluorescent spectrum of BSA in the lack and existence of NT in the PBS buffer (pH = 7.4) is displayed in Fig. 1 (Javaheri-Ghezeldizaj et al., 2020). BSA has an intense fluorescence emission peak of about 350 nm below the excitation wavelength of 290 nm, and after adding NT from 0 to 60 μM, a decline in fluorescence spectra was recorded without any significant change in the emission wavelength. This trend indicates that the innate fluorescence of BSA is quenching owing to interaction with NT (Javaheri-Ghezeldizaj et al., 2020). Quenching mechanisms can be identified by three factors: temperature, viscosity, and excitation state life. In the static quenching method (by complex formation), increasing the temperature reduces the stability of the base state complex, followed by a reduction of the quenching constant (K_SV), and therefore the formation of a ligand–protein complex with non-fluorescent properties (independent of diffusion) (Tantimongcolwat et al., 2019). In dynamic quenching, on the other hand, the absorption spectrum is not affected by extinction because it is effective only in the excitation mode of the quencher (Taheri et al., 2022). In dynamic quenching, high temperature causes quicker dissemination steady and progresses electron transfer, and therefore, K_SV tends to increase (Wani et al., 2018; Banu et al., 2021). The Stern-Volmer steady-state Eq. (2) was utilized to confirm the nature of the BSA fluorescence quenching mechanism due to the formation of the complex with NT (Javaheri-Ghezeldizaj et al., 2020).

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Fig. 1

Inherent fluorescence spectrum of BSA (2 mM) in the absence and presence of various NT concentrations (0, 10, 20, 30, 40, 50 and 60 μM) at 288, 293, 298, 303, and 310 K temperature.

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According to Eq. (2), F₀ and F show the intensity of BSA fluorescence in the lack and existence of NT (quencher). K_SV (Stern-Volmer constant), [Q] indicates the initial concentration of the quencher, k_q is the quenching rate of biomolecules of a protein (BSA) and $\tau$ ₀ is the lifespan of the exciting mood of the protein in the lack of any quencher (Maheri et al., 2022). K_SV values can be attained by the slope of the F₀/F plotting versus [Q]. The Ksv values are important from the viewpoint of fluorescence quenching. As revealed in Fig. 2A and Table 1, there is a powerful linear connection between F₀/F and [Q] for the Stern-Volmer diagram. Static and dynamic quenching can result in a Stern-Volmer linear diagram. The difference is that in hybrid mechanisms the diagram is nonlinear and this diagram shows an upward curvature. In general, with a raising temperature, the K_SV values decrease in the static quenching process but in the dynamic quenching process, temperature rising leads to a constant increase in diffusion and promotes electron transmission, so K_SV tends to increase (Shi et al., 2017; Mahmoudpour et al., 2020; Javaheri-Ghezeldizaj et al., 2021). K_SV and k_q values are shown in Table 1. Considering the K_SV values increase from 288 to 310 K (5.32 × 10³ to 10.0 × 10³) indicates that fluorescence quenching is more compatible with dynamic quenching instead of static quenching. However, at different temperatures, the k_q values are more than the maximum collision quenching constant (2.0 × 10¹⁰ Lmol⁻¹s⁻¹), signifying the occurrence of static quenching. Therefore, it can be inferred that NT reduced the fluorescence of BSA via a hybrid quenching mechanism (Sandhya et al., 2011; Shi et al., 2017; Nan et al., 2019; Javaheri-Ghezeldizaj et al., 2021).

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Fig. 2

A) The Stern–Volmer diagrams of the quenching of BSA fluorescence by NT at five different temperatures B) Modified Stern-Volmer plots for quenching of BSA upon interaction with NT at five different temperatures.

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Fluorescence quenching data were additionally evaluated by a modified Stern-Volmer Eq. (3) (Ali et al., 2023):

3.2 Binding constant and number of binding locations

If small molecules such as NT interact separately with a set of equivalent sites on biomacromolecules such as BSA, the possible binding stoichiometry (n) and the binding constant (K_b) of NT-BSA can be analyzed by the slop and intercept double logarithm regression curve utilizing the following equation (Bhojwani et al., 2023):

3.3 Analysis of thermodynamic parameters

Intramolecular interaction forces consist of electrostatic bonding, hydrogen bonding, van der Waals forces, and hydrophobic interactions that play a significant function in protein binding to various ligands. Enthalpy change (ΔH°), Gibbs free energy (ΔG°), and entropy change (ΔS°) are among the thermodynamic parameters, which help to identify the type of forces influencing the interaction between two molecules. Positive values of ΔH° and ΔS° represent the presence of hydrophobic interaction between two molecules and negative values of ΔH° and ΔS° are signs of van der Waals forces or hydrogen bonds occurring. A value of ΔG° less than zero indicates that a spontaneous interaction between NT and BSA occurs at the appropriate temperature. Finally, a negative sign for Δ ° indicates that the interaction of NT with BSA is enthalpy-driven. Thermodynamic parameters can be calculated by the following van’t Hoff and Gibbs free energy Eqs. (5) and (6):

According to Eq. (5), R is the universal gas constant (8.314 J mol⁻¹ K⁻¹) and T describes the experiential temperature. Van't Hoff diagrams for the interaction of BSA and NT are demonstrated in Fig. 3A. The values for ΔH°, ΔG°, and ΔS° at five temperatures are given in Table 1. The results revealed that the values of ΔS° and ΔH° are −237.6 J mol⁻¹ K⁻¹and −87.16 kJ mol⁻¹, respectively. Negative values of ΔH° and ΔS° confirmed that van der Waals forces, or hydrogen bonds, are the basic forces in the interaction of NT with BSA (Ross and Subramanian, 1981; Zhang et al., 2012; Wani et al., 2017; Wani et al., 2018; Wu et al., 2019; Javaheri-Ghezeldizaj et al., 2020).

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Fig. 3

A) The van't Hoff diagrams for the interaction of BSA with NT B) Absorption spectra of BSA (2 mM) before and after the addition of NT (0, 10, 20, 30, 40, and 50 μM). The red line showed the absorption spectra of NT.

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3.4 UV–Vis absorption spectroscopy

UV–Vis spectrum measurement is a procedure for analyzing structural changes and recognizing the formation of protein complexes upon interaction with small molecules, which is used in protein–ligand research. In this study, we evaluated the interaction of BSA with different amounts of NT using UV–Vis spectroscopy. Owing to the presence of aromatic amino acid residues (tryptophan, phenylalanine, and tyrosine residues) in the BSA structure, an innate absorption peak of about 210 (sharp peak) and 280 (weak peak) nm can be seen in Fig. 3B. By adding different amounts of NT (10 to 60 μM) to the BSA solution, the BSA adsorption spectra at 280 nm gradually increased. In general, abnormalities in the albumin absorption spectrum in the formation of the albumin ligand complex are evident. Based on this, it can be concluded that the change in the structure and microenvironment of BSA was owing to the interaction with NT and the formation of the BSA-NT complex (Hu et al., 2006; Banu et al., 2021).

3.5 Immobilization of BSA on CMD sensor chip

A schematic illustration of the BSA immobilization on the surface of a gold chip via amide linkage is shown in Fig. 4A (Fathi et al., 2019). As can be seen in the figure, the carboxylic groups on the surface of the CMD chip were activated using the EDC/NHS solution injected into the device. And in the next step, by injecting BSA into the device, a association was established between the active carboxyl groups on the surface of the CMD chip and BSA, and thus BSA was stabilized on the surface of the chip. In the last step, ethanolamine solution was used to block unreacted sites (Maleki et al., 2020). The sensorgram of these steps is shown in Fig. 4B. Measurements showed that at pH 7.4 the immobilization level of BSA on the CMD chip surface was acceptable (Taghipour et al., 2018).

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Fig. 4

A) Schematic plot of BSA immobilization by amine coupling. B) SPR sensorgram of BSA immobilization processes on a CMD chip.

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3.6 Kinetic parameters of BSA interaction with Natamycin

The reversible reaction of BSA-NT complex formation and separation can be shown by a simple Langmuir reaction: $$A+B\underset{K_d}{\stackrel{K_a}{⟷}}AB$$

To calculate the kinetic properties, it is necessary to calculate the k_a, k_d, and K_D for the binding of BSA to NT. The association constant is denoted by k_a, while the dissociation is denoted by k_d.

BSA sensorgrams in the existence and lack of various concentrations of NT (0.1–1.2 µM) at four temperatures (298, 303, 308, and 310 K) have been shown in Fig. 5 and its related data has been demonstrated in Table 2. The k_a of the BSA interaction with NT decreased when temperature increased while the k_d of the interaction was constant and therefore the values of K_D have been increased. The attained results demonstrated that the values of k_a play a major role in the variation of K_D values. Besides, increasing K_D values upon raising temperature indicated that BSA binding to NT decreased, which verified the attained data from the calculation of binding constants through a fluorimetry study.

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Fig. 5

The SPR Sensorgram of immobilized BSA interaction with various concentrations of NT as a food additive at four temperatures (A) 293 °K, (B) 298 °K, (C) 303 °K, and (D) 310 °K.

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3.7 Molecular docking results

One of the most widely used methods for studying the interaction of small molecules with biomolecules is the study of molecular binding. In the present study, the interaction of the NT and BSA biomolecule was investigated by applying of Auto Dock 4.2 package. The tertiary structure of BSA is classified into three binding domains (homologous), which are nominated as I, II, and III with subdomains A and B. The major ligand-binding region is located in some hydrophobic pockets in subdomain IIIA (Szymaszek et al., 2022).

In the present study, the blind docking study displayed that NT could bind to a pit that is located among IIIA (Sudlow I) and IB domains (Fig. 6A). Sudlow I is an important binding site on BSA that several big molecules enable to bind there due to the extended entrance gate. The importance of Sudlow I lies in its ability to bind a wide range of drugs, which makes it a useful element for drug delivery and development. Many drugs, including non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics, and anti-cancer agents, bind to Sudlow I with high affinity, leading to their sequestration in the plasma and reduced toxicity. Moreover, the binding of drugs to the Sudlow I site in BSA alters the pharmacokinetics and pharmacodynamics of the drugs. It affects the duration of drug action, the volume of distribution, the metabolism, and the elimination of the drug (Solanki et al., 2021).

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Fig. 6

A) Represents the binding site of NT on BSA in a cavity that is located among IIIA and IB. B) The detailed interaction between NT molecule and BSA on IIA and IB binding domains.

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To provide more details, another docking calculation was carried out with a particular focus on Site II. This site is known as an important position for the binding of many heterocyclic and aromatic compounds. Some studies also reported that Site II is an essential position for binding some fatty acids onto serum albumin (Tayeh et al., 2009; Belatik et al., 2012).

As indicated in Fig. 6B, Ser 109, Asp 111, Lys 114, Leu 115, Glu 424, and Arg 458 have an important role in NT-BSA interaction. The sign and magnitude of ΔS° and ΔH° indicated that the main significant interaction force for the binding between NT and BSA was van der Waals forces or hydrogen bond (Cheng, 2012). The binding was predicted to be −36.526 kJ mol⁻¹, which confirms the spontaneous binding of this ligand onto BSA without any requirement for external energy. As shown in Table 1, the thermodynamic study found the binding free energy (ΔG°C) of the NT-BSA complex to be −16.35 kJ mol⁻¹ at room temperature, but this varied from the molecular docking calculation. It is assumed that the theoretical research did not consider interstitial water molecules and buffer salts in the structure of BSA, which could have contributed to the association between NT and BSA molecules via the hydrogen bonding network (Diana et al., 1989; Abou-Zied and Al-Lawatia, 2011).