The compound under study – 2-(2-hydroxy-4-methoxybenzylidene)-1-(1H-benzimidazol-2-yl) hydrazine, was synthesized in a four-step reaction pathway (Argirova et al. 2021). o-Phenylenediamine, potassium hydroxide and carbon disulfide were used to obtain benzimidazole-2-thione which was further oxidized by alkaline solution of KMnO₄ to afford the corresponding 1H-benzimidazol-2-yl-sulfonic acid. As a next step a nucleophilic substitution of the sulfo group was carried out with hydrazine hydrate and finally the hydrazine derivative was converted in the target compound by condensation with 2-hydroxy-4-methoxy-benzaldehyde. Detailed description of the synthesis and product identification is reported in (Argirova et al. 2023).

A Scinco 2 South Korea spectrofluorimeter was used to measure fluorescence spectra. The slit widths were 2.5 nm for excitation and 10 nm for emission wavelengths in a 3 mL quartz cuvette with a 10 mm path length. 2H4MBBH–HSA fluorescence measurements were carried out by keeping the concentration of HSA fixed at 4 μM and those of 2H4MBBH were 10, 20, 30, 40, and 50 µM. Fluorescence spectra were recorded at two different temperatures of 15 and 25 °C in the spectral emission range 300–500 nm upon excitation at 283 nm (n = 5 replicates).

According to the literature, fluorescence spectroscopy is broadly utilized for exploring the interactions between drugs and proteins (Macii and Biver 2020). The inherent fluorescence of serum albumins shows up at 340 nm when excited at 280 nm, which is due to the presence of three aromatic l-amino residues (tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)). The intrinsic fluorescence of serum albumins is primarily contributed by the Trp and Tyr buildups since phenylalanine has an extremely low quantum yield. The fluorescence characteristics are remarkably sensitive to the microenvironment of the fluorescent residues or changes within the neighborhood environment of serum albumins, such as conformational transitions, biomolecular binding and denaturation (Santhamani and Sambandam 2013).

Fluorescence quenching diminishes the quantum yield of fluorescence from a fluorophore caused by an assortment of atomic interactions, such as ground-state complex formation, excited-state reactions, molecular improvements, energy transfer and collisional quenching. The distinctive instruments of quenching are more often than not constructed as either static quenching, or dynamic quenching (Lakowicz 2006).

For the purpose of elimination of the inner-filter effects, a method proposed by Lakowicz is shown in eq 1 (Lakowicz 2006):

F_cor=F_obs × e ^Aex+Aem2 (1)

where F_cor and F_obs represent the corrected and observed fluorescence intensities, respectively, whereas Aex and Aem denote the absorbance values at excitation and emission wavelengths, respectively. The corrected fluorescence was used for further analysis related to HSA fluorescence quenching.

To characterize further the fluorescence quenching mechanism of the 2H4MBBH-HSA system, the quenching experiments were conducted at two different temperatures, 15 and 25 °C. The fluorescence spectra of HSA in the presence of 2H4MBBH at different concentrations are shown in Fig. 2.

Addition of 2H4MBBH caused quenching of the HSA fluorescence at both temperatures. HSA exhibited a strong fluorescence emission peak at 338 nm upon excitation with wavelength of 283 nm. The fluorescence intensity of HSA showed a significant decrease with a well-expressed shift of the peak toward shorter wavelengths (from 338 to 329 nm), after the addition of 2H4MBBH. This shows that 2H4MBBH has apparently associated with HSA and quenched its intrinsic fluorescence, so that the micro-environment of the tryptophan residue in HSA has changed, producing an increment of hydrophobicity within the region of this residue. To resolve the fluorescence quenching mechanism, the well-known Stern-Volmer condition was utilized (Lakowicz 2006):

$\frac{F_0}{F}=1+K_{𝑆𝑉}[𝑄]=1+{𝐾}_{𝑞}{𝜏}_0[𝑄]$ (2)

where F₀ and F are the fluorescence intensities in the absence and presence of quencher, respectively, Q is the total concentration of the quencher (2H4MBBH), and K_SV is the Stern-Volmer quenching constant. Kq and τ₀ are quenching rate constant, and the average lifetime for the biomolecule without quencher, respectively. Since the fluorescence lifetime of the biopolymer was assume to be 10^-8 s (Lakowicz 2006; Lakowicz 1983), the quenching rate constant, Kq can be calculated using the following equation:

${𝐾}_q=\frac{{𝐾}_{sv}}{{𝜏}_0}$ (3)

The values for K_SV and Kq at the two temperatures are given in Table 1.

It is known that linear Stern-Volmer plots indicate a single type of quenching mechanism as predominant, either static or dynamic (Lakowicz 2006). The intensity changes are given in Stern–Volmer representation in Fig. 3. From the plots of F₀/F vs. Q the type of quenching can be selected, i.e., these plots can either correspond to static or dynamic quenching, or display a upward bend for mixed quenching types (Lakowicz 2006). In order to obtain values for the fluorescent quenching constant we used the Nedler-Mead simplex algorithm to fit a linear regression model to F₀/F and Q for the values of 2H4MBBH in the range 0–50 µM (Fig. 3).

The quenching constant Kq and the Stern–Volmer constant Ksv at different temperatures are usually used to identify the quenching mechanisms, static or dynamic. The former is caused by ground-state complex formation, the latter is due to the diffusion (Lakowicz 1983). For the static quenching mechanism, the quenching constant decreases with temperature increase (Lakowicz 2006). Moreover, in Table 1, the values of Kq at different temperatures were much higher than the limiting diffusion rate constant of the protein molecules (kd ≈ 2.0 × 1010 M−1·s−1) (Bhat et al. 2010), which revealed static quenching mechanism via forming protein-ligand complexes between 2H4MBBH and HSA complex.

For the static quenching process, the number of binding sites can be obtained by a double-logarithmic equation (Lakowicz 1973):

lg[(F0 − F)/F] = lgKa + nlg[Q] (4)

where F0 and F are the fluorescence intensities without and with the ligand, and Ka and n are the binding constant and the number of binding sites, respectively.

Based on this equation, the slope of the log((F0F)/F) two fold logarithmic regression curve versus the log[complex] (Fig. 4) was utilized to assess the number of binding sites (n).

The slope of the lines is the n value. If the value of n is equal to 1, it means that a strong binding exists between the protein and the drug. Our results suggest that inside the temperature range considered, the estimated number n of the 2H4MBBH-HSA complex is close to 1, demonstrating that HSA contains a single high affinity binding location for 2H4MBBH.

Ka is calculated to be roughly 10⁴, showing strong binding interactions between 2H4MBBH and HSA (Table 2).

It is additionally found that, as the temperature increases, the Ka value diminishes, suggesting that the stability of Mab–HSA complex diminishes with temperature increase. The estimate of Ka is critical to evaluate the attraction of the drug to plasma proteins. The binding of 2H4MBBH to HSA is of significance, because it determines the pharmacological activity of the medication. It is known that protein-binding may modify drug action in two diverse ways: by changing the medication effective plasma concentration at its location of activity, or by changing the rate at which the drug is dispensed with, hence changing the period of time for which viable concentrations are kept up.

The thermodynamic parameters provide remarkable data when examining the interaction between biomolecules. These parameters provide important data on the leading target interaction due to the high sensitivity of the binding characteristics to intrinsic and external components.

Binding forces involved in the interaction between serum albumin and ligands can be determined by calculating the thermodynamic parameters enthalpy (ΔH°) and entropy (ΔS°). These parameters give rich data on the interaction due to the high sensitivity of the apparent binding characteristics to intrinsic and extrinsic factors.

To better understand the binding between HSA and 2H4MBBH, the van’t Hoff Eq. (4) was used to calculate the thermodynamic enthalpy ΔH° and entropy ΔS° of the HSA and 2H4MBBH complex.

$In{K}_a=\frac{-\Delta H^0}{RT}+\frac{\Delta S^0}{T}$ (5)

where R is the universal gas constant (1.987 cal·K⁻¹·mol⁻¹) and T is the absolute temperature in degrees Kelvin.

To delineate the intermolecular forces existing between 2H4MBBH and HSA, a thermodynamic system was utilized and assessed at 15 and 25 °C. The standard binding free energy ΔG° is related to the binding constant Ka by the Gibbs relationship:

ΔG°=ΔH°–TΔS°=–RTlnK_a (6)

where K_a represent the binding constant at its corresponding temperature and R is the gas constant. ΔG° can be determined using van’t Hoff plot, where ΔH° is the slope and ΔS° the intercept.

Our results revealed negative thermodynamic parameters for the enthalpy ΔH° = – 48.47 kJ/M and entropy ΔS° = – 237.5 J.M^-1 of binding, and thus clearly emphasized that the interaction between 2H4MBBH and serum albumin is exothermic. Concurrent with the Ross theory (Ross and Subramanian 1981), negative values of ΔH° and ΔS° show that the binding may be enthalpy driven, whereas non-covalent attractive forces such as hydrogen holding and van der Waals dispersion forces in low dielectric media and protonation are basically responsible for the formation of a stable complex. Moreover, the binding of 2H4MBBH with HSA was spontaneous and enthalpy-driven process because |ΔH° |>|TΔS°|. On the other hand, if ΔG°>0, the process is spontaneous at low temperatures. The obtained results demonstrate a spontaneous binding of the drug to the protein molecule, represented by a positive sign of ΔG° = 19.67 kJ M^-1 and ΔG°= 22.037 kJ M^-1 at both temperatures, 15 and 25 °C, respectively [13].