Counteraction of lactose on the thermal stability and activity of α-chymotrypsin: thermodynamic, kinetic and docking studies

Abstract

Stabilized aqueous solutions of α-chymotrypsin have a therapeutic utility in the treatment of certain forms of asthma, bronchitis, rhinitis, sinusitis, as well as certain dermatological conditions such as leg ulcers and ringworm. The aim of the present study was to investigate how lactose could influence the structure, thermal stability and the activity of α-chymotrypsin. The influence of lactose on the structure and stability of α-chymotrypsin (α-Chy) was explored using thermal stability, fluorescence spectroscopy, circular dichroism (CD) kinetic studies and molecular docking. We have calculated the thermodynamic parameters for the transition temperature (T_m), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) to understand the stability of α-Chy. Lactose acted as an enhancer for the α-Chy stability, with varying efficacies and efficiencies. The results of the kinetic study displayed that the activity of α-Chy increased in the presence of lactose. The result reveals the ability of lactose to protect the native structural conformation of α-Chy. These results explicitly explain that stabilizing lactose is preferentially excluded from the surface of α-Chy, because water has a higher tendency toward favorable interactions with functional groups of α-Chy than with lactose. Fluorescence intensity changes showed static quenching during the lactose binding. The α-Chy fluorescence quenching suggested the more polar location of Trp residues. Near-UV and far-UV CD studies also proved the transfer of Trp, Phe and Tyr residues to a more flexible environment. Increasing of the α-Chy absorption in the presence of lactose was as a result of the formation of lactose–α-Chy complex. Molecular docking results revealed a negative value for the Gibbs free energy of the binding of lactose to α-Chy and the presence of one binding site. Docking study also revealed that hydrogen bond interactions dominated within the binding site.

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1. Introduction

Modification of the solvent environment is one of the best approaches to increase the stability and activity of enzymes. There are different approaches used to increase the stability and activity of enzymes. One of the simplest of this method is the addition of agents such as osmolytes directly into the protein solution. Organic osmolytes such as polyols are chemical chaperones that keep proteins from denaturation by supporting the maintenance of their naturally folded and functional states under various harmful conditions.¹ This study relates to the stabilization of chymotrypsin and more particularly to an aqueous solution of α-chymotrypsin, which is characterized by being substantially stable under ordinary conditions of commercial use.

Protein stability against denaturation, with little or no effect on their activity, increases when at near room temperature. Association of enzymes with inert supports can greatly affect their stability because of the change of the enzyme hydration level, dispersion in the medium or entrapment of the enzyme in a more active conformation.² Protein folding or protein–ligand interactions are affected by osmolytes such as lactose. These effects are mediated through solvation, the nonspecific interaction between the solution components. A few protective osmolytes are polyol and carbohydrate. They protect proteins against loss of activity as well as chemical and thermal denaturation.³ Effects of different osmolytes on protein stability and activity have been extensively investigated.^4,5 These studies have led to a greater understanding of the molecular thermodynamics of the protein–ligand interaction process.^6–8

It has long been known that α-Chy tends to become inactivated when dissolved in aqueous solutions. Such inactivation proceeds quite rapidly under high temperatures as well as denaturants, so it has therefore not been possible to manufacture and sell chymotrypsin in the form of an aqueous solution. It is the general object of this study offer a substantially stabilized aqueous solution of α-Chy–lactose, which is satisfactory for commercial manufacture and sale. Such aqueous solutions of α-chymotrypsin, have therapeutic utility in the treatment of certain forms of asthma, bronchitis, rhinitis, and sinusitis, as well as in the treatment of certain dermatological conditions such as leg ulcer and ringworm.⁹

The S1 family is one of the main families of serine proteases. α-Chymotrypsin (α-Chy) has 241 amino acids and comprises eight tryptophan residues. α-Chy is a model protein that we can work on the folding or unfolding process with the addition of co-solvents such as carbohydrates or polyols. Thus, α-Chy was chosen for the study of the thermodynamics and kinetics of the temperature-induced protein unfolding inhibited by lactose using different spectroscopic techniques at pH 8.0, molecular docking and molecular dynamics simulations.

Understanding the structure–function relationship is an important task in biochemical studies. The structural and functional information of α-Chy in the presence of osmolytes liquids are critical for understanding their metabolic role as well as their use in industry. In this study, the interaction between lactose and α-Chy was studied by multiple spectroscopic techniques as well as molecular docking and molecular dynamics simulations. The interaction mechanism between lactose and α-Chy was obtained through detailed analyses of the mechanism of quenching, binding constants, the specific binding site, thermodynamic parameters such as ΔS, ΔG, ΔH, T_m, and binding force. The effect of lactose on the conformation and activity of α-Chy was also investigated. We hope this study can not only clarify the binding mechanism of lactose with protein at a molecular level, but also offer a theoretical basis for adequately understanding its effect on protein function for industrial purposes and its action in vivo.

2. Materials and methods

2.1. Materials

α-Chymotrypsin (α-Chy) and N-benzoyl-L-tyrosyl-ethyl ester (BTEE) were purchased from Sigma and used without further purification. Lactose was purchased from Merk. All solutions were made in a 50 mM Tris–HCl buffer (pH 8.0).

2.2. Methods

2.2.1 UV-Vis absorption spectra. The UV-Visible absorption spectra were obtained using an Ultrospec 4000 Pharmacia UV-Vis Spectrophotometer equipped with a thermostatic cell holder. The concentration of α-Chy was 0.1 mg mL⁻¹. The system was baselined with Tris–HCl buffer and then α-Chy spectra were obtained upon the titration of spermine. Absorbance value changes were recorded at 280 nm. All studies were carried out in quartz cells containing 0.1 mg mL⁻¹ α-Chy and different concentrations of lactose solution. The resulting absorbance changes in the presence and absence of lactose were plotted versus lactose concentration.

2.2.2 Thermal stability of α-Chy. The thermal stability of free protein and protein–lactose complexes was investigated by monitoring the absorbance intensities at different temperatures, according to the two states equilibrium model N ↔ U. The UV-Vis spectrum of α-Chy at 280 nm was obtained with UV-Visible spectrophotometer (Ultrospec 4000 Pharmacia) in the presence and absence of various amounts of lactose. The system was the first base-lined with Tris–HCl buffer and then α-Chy spectra were obtained upon the titration of lactose. All studies were carried out in quartz cells, including 0.1 mg mL⁻¹ α-Chy and different concentrations of lactose. The scan rate was 1 °C min⁻¹ at 280 nm for the solutions of α-Chy (0.1 mg mL⁻¹) in the presence and absence of the lactose. The absorption data were plotted as a function of temperature. The melting temperature, T_m, which was defined as the temperature in which ΔG° was zero, was determined as the transition midpoint of the melting curve.

2.2.3 Fluorescence spectroscopy measurements. Steady-state fluorescence measurement studies were carried out using a fluorescence spectrophotometer (Shimadzu RF-5301) that equipped with a temperature adjustable cell holder. The emission spectrum was obtained from 290 to 450 nm in the presence and absence of different concentrations of lactose at temperatures of 15, 25 and 35 °C, with the excitation wavelength being at 280 nm. The slits were 3 nm for excitation and 5 nm for emission scans. Fluorescence spectra were obtained at each step of the temperature elevating processes. The solution was secured for 4 min before the scan to equilibrate the sample solution fully. Fluorescence of α-Chy was excited in 280 nm generated by a xenon arc lamp. Fluorescence spectra were obtained over the range of 290–450 nm. Measurements were carried out on the systems of α-Chy-buffer, α-Chy–lactose-buffer as a function of temperature. In addition, the buffer was collected as backgrounds in the same temperature region. All studies were performed in quartz cells containing 2 cm³ (0.1 mg mL⁻¹) α-Chy and different concentrations of lactose solution.

2.2.4 Circular dichroism. Circular dichroism (CD) spectra in the far-UV (190–260 nm) and near-UV (260–320 nm) regions were obtained by an AVIV 215 spectropolarimeter at 25 °C, using 1 mm-path cells for far-UV and 10 mm-path cuvettes for near-UV experiments. The protein concentration was 8 μM (0.2 mg mL⁻¹) and 16 μM (0.4 mg mL⁻¹) in far-UV and near-UV, respectively. The spectra were obtained after 4 min incubation with lactose to allow sample equilibration as stated above. The protein secondary structure was assessed by the CDNN program, version 2.1.0.223, using a network trained with 33 complex spectra as the reference set.¹⁰ Results of all the CD measurements are expressed as mean residue ellipticity ([θ]_λ) in degree cm² dmol⁻¹ at a given wavelength λ (nm). According to statistical methods implemented in the CD software of CDNN, secondary structure changes of α-Chy were determined in the absence and presence of various concentrations of lactose. It should be noted that each observed θ_λ of the protein was corrected for the contribution of the solvent.

2.2.5 Enzymatic activity assay. The catalytic activity of 37.5 μg mL⁻¹ α-Chy (E.C. 3.4.21.1; type II from bovine pancreas, Sigma) was determined by a UV-Vis spectrophotometer at 256 nm and 37 °C by the rate of BTEE (N-benzoyl-L-tyrosine ethyl ester; Sigma) hydrolysis in 50 mM Tris/HCl, pH 8, and in the absence and presence of 0–1 M lactose for steady-state kinetics. The kinetic parameters for α-Chy were determined at 37 °C by measuring the steady-state activity of the enzyme with BTEE in the absence and presence of lactose.

2.2.6 Molecular docking studies. In the present study, the three-dimensional structure of sugars (Table 1) was drawn via the Hyperchem software package¹¹ and minimized by molecular mechanics method via an MM⁺ force field. 1.34 Å resolution crystal structure of protein α-Chy (PDB ID 1YHP) were downloaded from protein data bank (PDB) (http://www.rcsb.org). This structure was selected due to its Bos Taurus origin, better resolution and co-crystallization with protein complex. Docking calculations were performed by a Autodock 4.0 software package.¹² All waters and hetero-atoms attached to the proteins were removed. Docking simulations were run using a Lamarckian Genetic algorithm (LGA). The grid points for Autogrid calculations were set to be 126 × 126 × 126 Å with the active site residues at the center of the grid box. The docking parameters were set to an LGA calculation of 10 000 runs. The energy evaluations were set to 1 500 000 and 27 000 generations. The Population size was set to 150 and the rate of gene mutation and the rate of gene crossover were set to 0.02 and 0.8, respectively. The obtained conformations were then summarized, collected and extracted by using Autodock Tools. The first and the last conformation were assessed from a 100-ranked set of each complex using the VMD-Visual Molecular Dynamics.

Table 1 Structure of sugars (lactose)

Ligand	Structure ligand	3D Structure ligand
Lactose

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3. Results and discussion

3.1. Absorption spectroscopy

UV-Vis absorption measurements are a very good method to investigate the structural change of a protein and to gain the information of the protein–ligand complex formation. Therefore, a study was made on the effect of the lactose concentration on the absorption spectrum of α-Chy. The UV-Vis absorption spectra of α-Chy in the presence and absence of lactose were also recorded to confirm the quenching mechanism. As can be observed in Fig. 1(a) and (b), there was a main absorbance peak: a absorbance peak around 280 nm, which represent the tryptophan residues of α-Chy.¹³ The intensity of this peak changed with the addition of lactose. The results are able to prove that the binding between lactose and α-Chy makes the protein skeleton structure changed.¹⁴ This was due to the formation of the ground state complex between α-Chy and lactose. It was likely that this complex had a higher molar extinction coefficient than that in the unabsorbed state, but it had the absorption maximum at the same position.^15–18 The environment of tryptophan residues was changed upon interaction with lactose, and the hydrophobicity of the microenvironment of tryptophan residues was changed.^19,20

Fig. 1 (a) UV-Vis spectra of α-Chy in the presence and absence of lactose (0–0.16 mM) at pH 8.0 and 298 K, and (b) absorbance at 280 nm in the presence and absence of lactose (0–0.16 mM).

3.2. Thermal stability of α-Chy

One of the most important elements in the description of the structural and molecular basis of biological function is thermodynamic information. Description of the global protein folding studies is by the Gibbs free energy change (ΔG) and understanding the protein stability is by the enthalpy change (ΔH) and transition temperature (T_m). The net stability of a protein is defined as the difference in the standard Gibbs free energy (ΔG°) between the native (folded) and denatured (unfolded) states.²¹ We can represent the equilibrium between these two states using a simple mechanism shown below:

F (folded) ↔ U (unfolded)

The net protein stability is defined as eqn (1):^21,22

In this equation, R is the gas constant and T is the absolute temperature. F_U is a fraction of the denatured protein and and denote the concentrations and the free energies of the unfolded and folded states, respectively. K is the equilibrium constant.²³ Thermodynamic parameters of standard free energy change (ΔG°) and T_m were estimated to appraise the feasibility and the exothermic nature of the adsorption process. A fraction of the unfolded form, F_U, was computed by normalizing denaturation curves and utilizing eqn (2):⁸

In this equation, Y_obs shows the observed variable parameter at a given denaturant concentration. Y_F and Y_U are the variable specifications of the folded and unfolded states, respectively; moreover, these values were acquired by the linear extrapolation of pre- and post-transition regions.²⁴ The resulting changes in F_U, in the presence and absence of lactose were plotted versus temperature (Fig. 2).

Fig. 2 The fraction unfolded of α-Chy in various concentrations of lactose at pH 8.0.

The Gibbs free-energy of unfolding, ΔG°, as a function of temperature for α-Chy in the presence and absence of lactose, is shown in Fig. 3. These results can be used to determine T_m at which ΔG° = 0. Our results also suggested that by increasing lactose concentrations, the curves were shifted to the upper temperatures (Fig. 3). α-Chy melting points at variable concentrations of lactose can be observed in Table 2. As indicated in Table 2, by increasing lactose concentrations, T_m of α-Chy increased. The results clearly assess the change of T_m values, which correspond to the transition of α-Chy to the unfolded state, as a function of concentration of lactose. It can be observed that lactose increases the T_m of α-Chy (Fig. 3). Temperature is one of the deactivating factors. When enzyme thermal stability improved, other denaturing factors are simultaneously more resistant. The addition of lactose to aqueous solutions of α-Chy-buffer strengthens the hydrophobic interactions between nonpolar amino acid residues, leading to protein rigidification.²

Fig. 3 The effect of lactose on ΔG° of α-Chy in different concentrations of lactose at pH 8.0.

Table 2 T_m changes of α-Chy at variable concentrations of lactose

Lactose (M)	Tm (K)
0.0	315.1
0.5	317.5
1.0	318
1.5	323

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In polyols such as lactose induced protein stability, there are two classes of interaction: direct and indirect interactions. Binding of polyols to proteins via hydrogen bonds is a direct interaction model that can stabilize the native state. In the indirect model, changing the structure and dynamics of water in the solution can enhance the hydrophobic interactions between pairs of hydrophobic groups and thereby stabilize the native state.³

3.3. Fluorescence spectroscopy

The fluorescence measurement is a useful technique to study tertiary structures and dynamics, the denaturation process, thermodynamic and ligand binding parameters and the mechanism in proteins Trp, Tyr, or Phe, because the intrinsic fluorescence of these residues is sensitive to the polarity of environments along the transition.^25,26 The intrinsic fluorescence of Trp residues is selected thanks to the intensity of emission and the high sensitivity of the Trp fluorophore to changes in the polarity of its microenvironments.^25,27,28

α-Chy is a globular protein containing eight tryptophan residues. The intrinsic fluorescence of α-Chy is mainly contributed by the tryptophan residue alone.^29,30 The changes in the emission spectra from Trp are related to the protein conformational transitions, the subunit association (apart from anion binding) or direct denaturation.³¹

At the excitation wavelength of 280 nm, the fluorescence spectra of α-Chy with varying concentrations of lactose are shown in Fig. 4(a) and (b). The fluorescence intensity of α-Chy regularly decreased with the increasing concentration of lactose, but no important shift of the emission maximum wavelength was observed, indicating that lactose interacted with α-Chy and quenched its intrinsic fluorescence. Moreover, the intrinsic fluorescence spectrum of the N-acetyl tryptophan demonstrated no significant shift (data not shown) in the presence of lactose. Therefore, a change in fluorescence of α-Chy due to the binding of lactose to α-Chy, would affect the microenvironment around Trp.

Fig. 4 (a): Fluorescence quenching of α-Chy, λ_exi = 280 nm, and λ_emi = 290–450 in the presence of different concentrations of lactose at pH 8.0 and 288, 298 and 308 K. (b) Fluorescence intensity in 333 nm and in the presence of different concentrations of lactose at the pH of 8.0 and 288, 298 and 308 K.

The fluorescence intensity of α-Chy decreased regularly with the increasing lactose concentration, which indicated that lactose binds to α-Chy. This indicated that the lactose has interacted with α-Chy, thereby changing the structure of this protein, especially the microenvironment of Trp residues; this, in turn, led to a less hydrophobic environment for the fluorescence fluorophore placed. In the hydrophobic environment (buried within the core of the protein), Trp has a high quantum yield and therefore a high fluorescence intensity was observed. In contrast, in the hydrophilic environment (exposed to solvent) their quantum yields decreased, in which a low fluorescence intensity was observed.³²

3.4. Mode of fluorescence quenching

Dynamic and static quenching are different mechanisms of fluorescence quenching. A process that the fluorophore and the quencher (ligand) come into contact during the lifetime of the excited state is dynamic quenching. Static quenching refers to fluorophore–quencher complex formation. Dynamic or static quenching can be recognized by their different temperature dependencies.³³

By measuring the intrinsic fluorescence quenching of α-Chy, the accessibility of the quenchers to the fluorophore groups of α-Chy can be estimated. This information can help us to predict the binding mechanism of lactose to α-Chy. Changes in environment and structure of the protein can be investigated by the fluorescence measurements.¹⁹ The quenching mechanism determined by the Stern–Volmer equation^34–39 (eqn (3)):

where F₀ and F are the fluorescence intensities of α-Chy in the absence and presence of lactose (quencher), respectively; [Q] is the concentration of the quencher (ligand); and K_sv is the Stern–Volmer quenching constant and determined by liner regression of Stern–Volmer equation, K_sv is described by the following equation⁴⁰ (eqn (4)).

K_sv = k_qτ₀ (4)

where k_q is the quenching rate constant of the biological macromolecule and τ₀ is the fluorescence average lifetime of the fluorophore (in this case Trp), which has been reported for the Trp residues of α-Chy at the neutral pH as 2.96 × 10⁻⁹ s.^14,41 Eqn (3) was applied to determine K_sv by the linear regression of a plot of F₀/F against the concentration of lactose. F₀ and F are the steady state fluorescence intensities of α-Chy at 333 nm before and after the addition of quencher (lactose), respectively. A plot of F₀/F versus [Q] yields an intercept of one on the y-axis and a slope equal to K_sv. As shown in Fig. 5. Stern–Volmer plots were plotted based on eqn (4). The solid lines in Fig. 5 show the lines of the best fit of the experimental data to the Stern–Volmer equation. A linear Stern–Volmer plot is commonly indicative of a single class of fluorophore, all equally accessible to the quencher. Recognition of static and dynamic quenching can be evaluated by the effect of temperature and viscosity, or preferably, by fluorescence lifetime measurements. Higher temperatures result in faster diffusions and therefore larger amounts of collisional quenching. Higher temperatures will also typically result in the dissociation of weakly bound complexes and therefore smaller amounts of static quenching. The increase of K_sv with temperature indicated dynamic quenching. In Table 3, the binding constant was obtained from the Stern–Volmer method and listed for lactose with α-Chy. The k_q value was less than 2.0 × 10¹⁰ L mol⁻¹ s⁻¹. This also indicated that the quenching of α-Chy fluorescence by lactose is static quenching (Fig. 5). As we all know, dynamic quenching only affects the excited state of fluorophore, but the static quenching induces the change of the absorption spectrum of the fluorophore, so the result again emphasized that the quenching mechanism was static quenching initiated by the formation of the ground state lactose–α-Chy complex.⁴² Osmolytes tend to stabilize the native structure of proteins. Moreover, this carbohydrate increases the water structure and form a hydration layer with water molecules. During this period, the disulfide bonds and a surrounding network of α-Chy also formed a hydration layer with the water molecules. Obviously, α-Chy cannot interact with the hydration layer around the osmolytes.³

Fig. 5 Stern–Volmer plots for the quenching of α-Chy by lactose at 288, 398 and 308 K. λ_exi = 280 nm, λ_emi = 290–450 nm, and pH 8.0.

Table 3 Stern–Volmer quenching constants of the complex of α-Chy with lactose at pH 8

T (K)	Ksv (L mol^−1)	kq (L mol^−1 S^−1)	R^2
308	64.32 × 10^−2	21.79 × 10^7	0.9835
298	79.69 × 10^−2	26.92 × 10^7	0.9893
288	97.21 × 10^−2	32.84 × 10^7	0.9877

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3.5. Calculation of binding parameters

For the static quenching interaction, when small ligand binds independently to a set of equivalent sites on a protein, the binding constant (K) and the number of binding sites (n) can be evaluated by the following equation:^27,43

The curve of the double logarithm regression of log(F₀ − F)/F versus log[Q] for α-Chy–lactose complex at different temperatures has been shown in Fig. 6. The K and n values have also been listed in Table 4. According to eqn (5), the slope of the plot of static quenching of log[(F₀ − F)/F] vs. log[Q] reflects the binding number.^34,44 It was found that K decreased with increasing temperature. The value of n was approximately equal to 1, indicating that there was one binding site in α-Chy for lactose during their interaction.

Fig. 6 The plot of log[(F₀ − F)/F] vs. log[Q] at 288, 398 and 308 K for α-Chy with lactose, λ_exi = 280 nm, λ_emi = 290–450 nm, and pH 8.0.

Table 4 Binging and thermodynamic parameters of α-Chy–lactose interaction at pH 8.0 and three different temperatures

T (K)	K (×10^−2 M^−1)	n	R^2	ΔH° (kJ mol^−1)	ΔS° (kJ mol^−1 K^−1)	ΔG° (kJ mol^−1)
288	113.52	1.19	0.9977	−3490.13	−11.04	−310.61
298	108.86	1.26	0.9877	−3490.13	−11.04	−200.21
308	103.25	1.41	0.9969	−3490.13	−11.04	−89.81

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3.6. Determination of thermodynamic parameters

The dependence of binding constants on temperature shows that a thermodynamic process is responsible for the formation of the complex (α-Chy–lactose). This dependence was therefore analyzed to better characterize the forces acting between lactose and α-Chy.⁴⁵ Essentially, there are four types of noncovalent interactions that could play an important role in ligands binding to α-Chy. They are hydrogen bond, electrostatic and hydrophobic force, and also van der Waals interactions.^43,46 Stabilizing ligands bound to proteins: hydrogen bonds, electrostatic forces, van der Waals forces and hydrophobic interactions.⁴⁴

The sign and magnitude of the thermodynamic parameters (ΔH° and ΔS°) are important information for confirming the main forces involved in the binding reaction.¹³ The temperatures chosen were 288, 298 and 308 K at which α-Chy did not undergo any structural degradation and denaturation. If the enthalpies change (ΔH°) does not vary remarkably over the temperature range studied, its value and that of ΔS° can be evaluated from the Van't Hoff equation:^47,48

ΔG° = ΔH° − TΔS° (7)

where K is the constant of binding. The ln K versus 1/T plot (Fig. 7) enabled the determination of ΔH° and ΔS° for the binding process. The values of K, ΔH°, ΔS° and ΔG° were summarized in Table 4. The negative value of ΔG° displayed that the interaction process is spontaneous. The acting forces between proteins and ligands were classified on the base of the magnitude and the sign of thermodynamic parameters into four groups of non-covalent interactions: (a) ΔH° > 0 and ΔS° > 0, hydrophobic interactions; (b) ΔH° < 0 and ΔS° > 0, van der Waals force; (c) ΔH° < 0 and ΔS° < 0, hydrogen bond and van der Waals interactions and (d) ΔH° < 0 and ΔS° > 0 electrostatic interactions.^44,46 For α-Chy–lactose complexes, the main source of ΔG° value was derived from a large contribution of ΔH° term with a little contribution from ΔS° factor. Therefore, we inferred that hydrogen bond and van der Waals interactions might play a major role in the interaction of lactose with α-Chy.

Fig. 7 Van't Hoff plot of α-Chy by lactose. The ln K versus 1/T plot.

3.7. Circular dichroism spectra

3.7.1 Far-UV CD. Far CD spectroscopy is one of the most widely utilized techniques for detecting the secondary structure changes of α-Chy when interacting with ligands, thereby making it possible to quantify conformational modifications in the 3D structure.¹⁴ The analysis of far-UV CD spectra (190–260 nm) could be used to assess the content of different secondary structure elements in proteins. The CD spectrum of α-Chy in buffer had a minimum at ≈202−205 nm (depending on the pH value) and no positive band.^31,49 By using it, the fractions contents of different secondary structures of α-Chy in the absence and presence of lactose were calculated using the software package CDNN. The far-UV CD spectra of α-Chy and α-Chy–lactose mixture is shown in Fig. 8. α-Chy was folded into two domains with very little α-helix content and wild regions of anti-parallel β-sheets, along with two inter-chain and three intra-chain disulphide bonds.⁵⁰ Spectral deconvolution of α-Chy spectrum in the buffer showed a low decrease of α-helix by 2% and an increase of β structures by about 12%. In the conditions used here, the far-UV CD spectrum of α-Chy showed a negative band in the 205 nm region (Fig. 8).

Fig. 8 Far-UV CD spectra of α-Chy in the presence and absence of lactose at pH 8.0. The Y-axis is the mean-residue ellipticity with the unit of degree cm² dmol⁻¹.

By CD software of CDNN, secondary structure changes of α-Chy were determined in the absence and presence of various concentrations of lactose. In the presence of denaturants or higher temperatures, α-Chy unfolds and value of the initial secondary structures, such as α-helices and β-sheets, change to new value of secondary structures, or other type of secondary structures such as discrete turns or coils. In the carbohydrate solutions, the secondary structures of the protein are protected and the native structure of the protein is largely preserved. Carbohydrates are compounds with some hydroxyl groups, so these molecules may directly interact with protein through H-bonds.³ Lactose shows clear improvements in the β-structure. This indicates that lactose are stabilizing additives because they maintain the solvophobic interactions essential for the native state and preserve the water shell or cage around the α-Chy structure.

3.7.2 Near-UV CD. Near-UV CD (240–320 nm wavelengths) was also used to assure that the observed effects were related to α-Chy conformational changes. In this technique, the spectrum of a protein is essentially a contribution of Tyr and Trp residues and disulphide bonds that can be affected by the flexibility and the number of aromatic side chains. This technique can be very useful to study changes by external factors on tertiary structures caused.⁵¹ The near-UV CD spectra of the enzyme indicated that the α-Chy had a particular tertiary structure in each of the solvents with different concentrations of lactose.

The near-UV CD spectrum of α-Chy (Fig. 9) in the buffer displayed the contributions of Tyr and Trp residues responsible for the peaks and shoulders between 270 and 305 nm, and those of the Phe residues, which strongly contributes to bands in the 258–270 nm region.⁵² Therefore, the disappearance of these bands suggested an enhanced flexibility of peptide chains around the aromatic residues due to a partial unfolding of the proteins. The CD spectrum in the near-UV region was very complicated, since the microenvironment for each amino acid in a protein is different. Nevertheless, the asymmetry of the microenvironments was lost when the protein was unfolded, and the corresponding diminishes in the near-UV CD signals reflected the degree of tertiary structure loss around the aromatic chromophores.³¹

Fig. 9 Near-UV CD spectra of α-Chy in the presence and absence of lactose at pH 8.0. The Y-axis is the mean-residue ellipticity with the unit of degree cm² dmol⁻¹.

The results of the α-Chy structure in lactose showed maxima at 280–300 nm, which can be best assigned to Trp residues. The intensity of this band decreased when the aromatic residues were away from each other, i.e., the native structure of the protein was not preserved in the lactose solution. The ellipticity values in the near UV-CD region were changed with a decrease in the peak intensity in the presence of lactose. When lactose was added to the solution of the α-Chy, a gradual negative decline in the molar ellipticity values was observed. This difference in ellipticity can be attributed to the unfolding of the α-Chy. Therefore, these changes suggest that there is an enhanced flexibility of the peptide chain around the aromatic residues due to the partial unfolding of α-Chy. The analysis of near CD results has been displayed in Fig. 9. As can be observed in Fig. 9, α-Chy showed the change in tertiary structure at different concentration of lactose. After 5 min of incubation, lactose changes occurred in the 270–305 nm regions, thereby indicating a conformational change that led to a more flexible environment near the aromatic residues.

The intrinsic fluorescence only monitored the changes in some of the Trp residues, while the near-UV CD had contributions of all the aromatic residues, not only tryptophans.⁵² It is reasonable to assume that the spectral changes observed by intrinsic fluorescence, UV-Vis and near-UV CD were related to the changes in α-Chy conformation upon binding to lactose.

Entropy is an important driving force in protein folding. When folded, the α-Chy has a much lower degree of freedom, but sequestration of hydrophobic group's releases bound water molecules, so the entropy of the solvent is increased and the water becomes more structured in lactose solutions.⁵³ Therefore, the entropy of water decreases, leading to a larger transfer free energy. In the unfolded state of α-Chy, the solvophobic effect of lactose is larger than that on the folded state, so the native α-Chy more stable in a lactose solution than in a tris buffer.³

3.8. Enzymatic activity of α-chymotrypsin

The enzymes structure and conformational dynamics are influenced by both the temperature and the ligand. However, temperatures that are too low or too high denature proteins, thereby deactivating their function. Moreover, co-solvents can stabilize or destabilize the conformation of proteins and therefore, influence their enzymatic activity. Addition of lactose to a α-Chy solution stabilizes the enzyme by increasing the melting temperature, T_m. To find out whether the lactose could cause any alternation in the enzyme activity of the folding transition state of α-Chy, we further performed enzyme activity experiments. We tested the α-Chy activity in the presence of lactose using a UV-Vis spectrophotometer. The obtained results have been represented and displayed in Fig. 10 as the double reciprocal plot of line Weaver–Burk. The enzymatic activities of α-Chy in the presence and absence of lactose was measured at 37 °C and the pH 8.0 (Fig. 10). It can be observed from Fig. 10 and Table 5 that the activity of α-Chy was increased by increasing the concentration of lactose. Lactose is a non-essential activator for α-Chy.⁵⁴ The binding of lactose may change the micro-environment of the catalytic triad-His57, Asp102, and Ser195 for the intruding molecule in the their vicinity, from the overall perspective.⁵⁵ Consequently, according to the results, it was found that T_m of α-Chy and enzyme activity was increased with the addition of lactose concentrations. As a result, lactose could be regarded as an activator and stabilizer for trypsin.

Fig. 10 Line Weaver–Burk plot of α-Chy at various lactose concentrations (M) at 37 °C, pH 8.0.

Table 5 Parameters of α-Chy activity in the presence of lactose

[Lactose] mM	Km (mM)	Vmax (mM s^−1)	kcat (s^−1)	kcat/Km (mM^−1 s^−1)
0	1.06	0.041	26.0 × 10^3	24.5 × 10^3
0.1	1.52	0.070	47.1 × 10^3	31.0 × 10^3
0.3	2.73	0.1481	94.8 × 10^3	34.7 × 10^3

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3.9. Molecular modelling study

3.9.1 Molecular docking studies. The results of docking could help to identify the potential localization of binding sites and show a unique site of interaction between α-Chy and lactose (Table 6). Moreover, Fig. 11 shows H-bond and hydrophobic analysis of docking poses by a Ligplot plus tool for lactose ligands in α-Chy and Fig. 12 displays the interaction of lactose with α-Chy. The three-dimensional (3D) structure of α-Chy was obtained from the protein database, PDB. Autodock needed receptor and ligand representations in a file format called pdbqt, a modified protein data bank. For docking the lactose in to the binding site of the α-Chy, we used AutoDock 4.2 Software. The search algorithm was used in the AutoGrid program based on defining all active molecules and generating the grid parameter files. Then, the scoring algorithm in Autodock program was used for the binding conformation of the ligands. By using a Lamarckian genetic algorithm (LGA), a hundred runs of docking were performed. Ligplot plus was used to analyze the docking poses for hydrogen bonding and hydrophobic bonding. A single binding site could be deduced from the analysis of spectroscopic studies. The Gibbs free energy (ΔG°) of lactose binding to α-Chy was equal to −4.41 kJ mol⁻¹. Nevertheless, the negative sign of ΔG° corresponded well with the experimental results, thereby indicating that the binding process was spontaneous. The results obtained from the docking study are in agreement with those from the fluorescence spectroscopy measurements in which the number of binding sites is 1. In addition, both molecular docking and CD spectroscopy show that lactose does not induce any significant changes in α-Chy structure. Moreover, the molecular docking results show that the quenching of α-Chy is for change of Trp environment. The Gibbs free energy value (ΔG°) of binding calculated from docking was −4.41 kJ mol⁻¹, what diverges from other experimental results. However, this negative value indicates that the binding process is spontaneous. A possible explanation may be that the pdb structure of the crystallized α-Chy differs from its structure in aqueous medium as used in this study.

Table 6 Docked results with interacting residues after 100 runs of docking

Lowest binding energy (kJ mol^−1)	Inhibition constant (298.15 K), pKi	Final intermolecular energy (kcal mol^−1)	vdW^a + H bond + desolv energy (kcal mol^−1)	Electrostatic energy (kcal mol^−1)	Final total internal energy (kcal mol^−1)	Interaction bonds
						Hydrogen-bonding	Hydrophobic-bonding
a van der Waals.
−4.41	920.94	−7.72	−7.41	−0.38	−6.48	Ser217, Ser221, Thr224, Lys175, Ser218	Thr219, Tyr171, Trp172, Trp215

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Fig. 11 H-bond (black) and hydrophobic (red) analysis of docking poses by Ligplot plus tool for Lactose ligands in α-Chy.

Fig. 12 The superimposition of the docked lactose into protein α-Chy.

3.9.2 Accessible surface area calculations. The ASA values for the Trp residues of α-Chy and the complex near the binding sites and active site are presented in Tables 7 and 8. It is found that the value of ASA for the active site residues and Gly193 increased in the binding interaction, which is much more than α-Chy structure. This shows that α-Chy can strongly interact with lactose. The residues His57, Ser195 and Gly193 of α-Chy suffer a considerable change in ASA due to the hydrogen and hydrophobic interactions with lactose, which is what we expected (Fig. 11). The ASA calculations also show that Trp residues obtained a substantial amount of ASA in the binding interaction (Table 7).

Table 7 ASA for the residues of α-Chy and lactose–α-Chy complex in Ų

Residues	ASA for α-Chy	ASA for complex	ΔASA	Change of ASA (%)
Trp27	6.12	3.24	2.88	−47.05
Trp29	1.05	0.19	0.86	−81.90
Trp51	3.02	5.81	2.79	+92.38
Trp141	16.64	28.29	11.45	+68.81
Trp172	33.42	38.43	5.01	+14.99
Trp207	26.13	3.87	22.26	−85.18
Trp215	65.56	45.96	19.6	−29.89
Trp237	54.29	44.59	9.7	−17.86

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Table 8 ASA for the active site residues of α-Chy and lactose–α-Chy complex in Ų

Residues	ASA for α-Chy	ASA for complex	ΔASA	Change of ASA (%)
His57	69.16	59.22	9.94	−14.37
Asp102	0.70	0	0.70	−100
Ser195	24.96	14.11	10.85	−43.46
Ser214	3.34	0	3.34	−100
Gly193	33.06	48.208	15.148	+45.81

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In addition, the increase of ASA for Trp51 of α-Chy is 92.38%, which suggests a great change of micro-region. The result is colloquial with the conclusions of the fluorescence spectrum. Therefore, this fact also provides a good basis to explain the efficient fluorescence quenching of α-Chy in the presence of lactose. ASA calculations also prove that hydrogen bonds play an important role in the binding of lactose to α-Chy. This is in agreement with the thermodynamic and molecular docking analysis. Docking studies also showed that some of Trp residues had an increase in ASA, relative to the polar solvent. Therefore, quenching of the protein's fluorescence could lead to increase in ASA.

4. Conclusion

In the present study, spectroscopy and molecular docking were applied to characterize the interactions between α-Chy and lactose. In all cases, molecular docking results are in agreement with spectroscopic results. The experimental results indicated that the microenvironments of the tryptophan residue of α-Chy changed with lactose, and the quenching mechanism of α-Chy by lactose was a static quenching procedure. In addition, from the thermodynamic parameters, there is one binding site. Molecular docking also showed one binding site. The same results from accessible surface area calculations and fluorescence emission measurements confirm this quenching. ΔG° values determined at 288, 298 and 308 K were negative. Molecular docking as well as the experimental results yielded the negative values of ΔG° and revealed that the interaction process is spontaneous. Kinetic results and thermal studies also demonstrated that by increasing the concentration of lactose, the stability and activity of the enzyme was enhanced. The docking study also indicated that lactose was absorbed on the surface of the enzyme, leading to an increase in the thermal stability. Molecular docking combined with spectroscopy is very pertinent in obtaining credible results of binding studies and this combination is also helpful in determining the mechanism of interaction. Therefore, we believe that this study has highlighted a method that could offer a new research approach in biological systems.

Acknowledgements

The study was financially supported by Shahrekord University, Iran.

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