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. 2023 May 22;8(22):19853–19861. doi: 10.1021/acsomega.3c01755

Interaction Mechanism between α-Lactalbumin and Caffeic Acid: A Multispectroscopic and Molecular Docking Study

Nasser Abdulatif Al-Shabib †, Javed Masood Khan †,*, Abdulaziz M Al-Amri ‡, Ajamaluddin Malik ‡, Fohad Mabood Husain †, Prerna Sharma §, Arnold Emerson ∥, Vijay Kumar ⊥, Priyankar Sen #,*
PMCID: PMC10249380  PMID: 37305235

Abstract

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Caffeic acid (CA) is a phenolic acid found in a variety of foods. In this study, the interaction mechanism between α-lactalbumin (ALA) and CA was explored with the use of spectroscopic and computational techniques. The Stern–Volmer quenching constant data suggest a static mode of quenching between CA and ALA, depicting a gradual decrease in quenching constants with temperature rise. The binding constant, Gibbs free energy, enthalpy, and entropy values at 288, 298, and 310 K were calculated, and the obtained values suggest that the reaction is spontaneous and exothermic. Both in vitro and in silico studies show that hydrogen bonding is the dominant force in the CA-ALA interaction. Ser112 and Lys108 of ALA are predicted to form three hydrogen bonds with CA. The UV–visible spectroscopy measurements demonstrated that the absorbance peak A280nm increased after addition of CA due to conformational change. The secondary structure of ALA was also slightly modified due to CA interaction. The circular dichroism (CD) studies showed that ALA gains more α-helical structure in response to increasing concentration of CA. The surface hydrophobicity of ALA is not changed in the presence of ethanol and CA. The present findings shown herein are helpful in understanding the binding mechanism of CA with whey proteins for the dairy processing industry and food nutrition security.

Introduction

Caffeic acid (CA) is a yellow-colored organic compound. CA is a polyphenol commonly found in a variety of foods.1 Different foods and beverages have different CA concentrations, for example, brewed coffee and red wine have 0.13 mg and 2.0 mg/100 mL, respectively.2 CA is abundantly found in herbs of the mint family, especially thyme, sage, and spearmint (around 20.0 mg/100 mL).3 CA has been shown to have antibacterial, antioxidant, antidiabetic, hepatoprotective, anticancer, and antiviral activities in vitro and in vivo.4−6 CA formed a complex with whey protein isolate (WPI) and attenuated oral sensitization in C3H/HeJ mice against WPI.7

α-Lactalbumin (ALA) is a whey protein, and its percentage of occurrence in milk is 3.8% of total milk proteins. It is a single-chain polypeptide, its molecular weight is 14.2 kDa, and its 3D structure is very similar to the 3D structure of lysozyme. ALA has two domains, a larger α-domain and two short domains.8 ALA binds strongly with Ca2+ and is also known as a model Ca2+ binding protein. ALA helps in lactose biosynthesis in the lactating mammary gland. ALA has also shown some other biological activities, such as apoptosis in tumor cells and bactericidal activity.9

Polyphenol-protein interactions have received much attention in recent years. Whey proteins can also transport hydrophobic molecules. Whey proteins interact with polyphenols noncovalently and serve as a delivery vehicle. Several reports have already shown the interaction of polyphenols with whole milk proteins [casein, bovine serum albumin (BSA), lysozyme, lactoferrin, β-lactoglobulin, and α-lactalbumin], whey proteins, and specific individual milk proteins.10 It was also found that the phenolic compound possesses an interacting property with ALA.

Phenolic acids found in food show good binding affinity with ALA.11,12 The antioxidant property of ALA is shown to be affected due to interaction with polyphenol, that is, epigallocatechin gallate.13 Curcumin is one of the well-known polyphenols that has shown strong interaction with serum albumin and whey proteins.14 Another polyphenol, that is, resveratrol, is known to bind with β-lactoglobulin; its secondary structure is unchanged, but it affects the tertiary structure.15 CA has also been found to bind with human serum albumin (HSA), BSA, and lysozyme.16,17 On BSA, it binds competitively at subdomains IIA and IIIA, while on HSA, it binds at IA. In lysozyme, the binding inhibits enzyme activity and increases its thermal stability.18 Many studies proposed that most polyphenols bind to the protein, and their interactions are primarily governed by hydrophobic interactions and hydrogen bonding.17 It will be necessary to discover the interactions between bioactive compounds/drugs and carrier proteins to comprehend the mechanism of transport of these compounds/drugs. Milk proteins can act as a natural carrier for transporting numerous bioactive molecules and vital micronutrients. As we know, ALA is known as a carrier protein, and exploring and deciphering the interactions between CA and ALA will be an exciting research topic. ALA has been found to have a strong binding affinity toward stearic acid, palmitic acid, and oleic acid (OA).19 ALA, which is nonapoptotic at its native state, binds to OA to form an apoptotic complex called HAMLET (Human Alpha-lactalbumin Made Lethal to Tumor cells) or BAMLET in the case of bovine ALA. Thus, it is important to understand the affinity of ALA toward other fatty acids or polyphenolic acids, so that such complexes could be tested for their toxicity on cells.20

The current research deals with CA’s interactions with ALA at a molecular level, which have been examined using several biophysical techniques. The interaction of whey proteins with biologically active compounds and molecules is an important area of study in dairy science. This type of research will aid in preserving the nutritional value. Therefore, the current work has significance and potential application in the food industry.

Materials and Methods

Materials

Bovine ALA, CA, and 1-anilino naphthalene sulfonate (ANS) were purchased from Sigma Chemical Company (St. Louis, Missouri, United States). Other reagents used were of analytical grade.

Methods

Solution Preparation

ALA was dissolved in a pH 7.4 phosphate buffer (20 mM). The ALA solution was clear and filtered by a syringe filter. Later on, the ALA concentrations were measured at 280 nm (A280) with an extinction coefficient of 28,540 M–1 cm–1.21 Next, CA powder was dissolved in ethanol, and the stock concentration was 3.0 mM. Later, the stocks were diluted around five times in the same buffer, and the working concentrations were 600 μM.

Fluorescence Spectroscopy

Intrinsic fluorescence was measured on a Cary Eclipse spectrofluorometer equipped with a Peltier temperature controller (Agilent Technologies, Inc., Santa Clara, CA, USA) using a Xenon flash lamp. The intrinsic fluorescence measurements were conducted according to previously published work with slight modifications.21 Intrinsic fluorescence was measured at temperatures of 288, 298, and 310 K. The ALA (3.0 μM) solution in a 3.0 mL (in 1 cm pathlength cuvette) cuvette was titrated using 3.0 μL of CA (to achieve 0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, and 9.0 μM CA) with stepwise addition from the stock of 600 μM CA and a magnetic stirrer-mixed sample solution. The spectra were scanned in the 300–450 nm range after excitation at 280 nm. The excitation and emission slit widths were fixed at 5.0 and 10.0 nm, respectively. The details of fluorescence quenching and thermodynamic data calculations are given in the supplementary file.

In order to minimize inner filter effects, corrections to the fluorescence measurements were done following a previous study.22

For the extrinsic fluorescence experiment, the ALA samples treated with 1% EtOH and 9.0 μM CA were incubated with 60 mM ANS for 90 min at room temperature in the dark. The samples were excited at 380 nm, and the emission spectra were recorded in the range of 400–600 nm. The emission and excitation slit widths were set at 5 nm.

Synchronous Spectra Measurements

A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to measure the synchronous fluorescence spectra in a quartz cuvette with a 1 cm path length and 3 mL volume. For synchronous fluorescence measurements, the parameters of Δλ = 15 and 60 nm and the scanning range of 260–310 and 250–320 nm were used, respectively. The ALA concentrations were fixed as 3.0 μM in the measurements.

UV–vis Absorption Measurement

The UV absorption spectra of ALA (15.0 μM) without and with CA at concentrations of 0, 30, 45, and 60 M were measured on a Cary 60 spectrophotometer equipped with a Peltier temperature controller (Agilent Technologies, Inc., Santa Clara, CA, USA). The protein spectra with and without CA were recorded from 250 to 400 nm. The absorption spectra of various concentrations of CA at pH 7.4 were also scanned. The blank was subtracted.

Circular Dichroism and Secondary Structure Calculation

Far-UV circular dichroism (CD) spectra of ALA/CA samples were recorded on a ChirascanPlus (Applied Photophysics Limited, Leatherhead, Surrey, United Kingdom) instrument at room temperature in the 200–250 nm wavelength range. ALA concentration was 15.0 μM, while the CA concentration was 1, 4.0, and 7.0 μM. The samples were prepared in a pH 7.4 sodium phosphate buffer (20 mM). Each spectrum was acquired two times, and the results were averaged and expressed in mdeg.

Molecular Docking and Simulation

The crystal structure of ALA is taken from the protein data bank using the pdbID:1F6S.23 In addition, we used the PubChem database to retrieve the 3D structure of the ligand CA.24 Then, the molecular docking was performed using Autodock 4.2,25 followed by molecular dynamic (MD) simulation. Generally, the docking conformation with the lowest binding free energy is considered the best. However, one should also check other criteria; RMSD (root-mean-square deviation) and the number of interacting residues/hydrogen bonding are very important. We used the above criteria to select the best docking conformation. The MD simulation calculates the time-dependent behavior of a biomolecular system. It thus provides detailed information on the fluctuations and conformational changes of proteins as a function of time.26 MD simulation was carried out for the protein–ligand complex using GROMACS 2018.4 simulation package.27 First, the protein structure was parameterized using the AMBERff99SB force field.28 Then, after energy minimization, the system was equilibrated initially with an NVT ensemble at 310 K, followed by an NPT ensemble for 1 ns till the system was stabilized. Finally, a 100 ns simulation was carried out, and the trajectories were analyzed using the Xmgrace tool.29

Results and Discussion

Intrinsic Fluorescence Measurements

Fluorescence spectroscopy is a commonly used technique to study the binding of proteins with a variety of ligands such as polyphenols, food additives, and surfactants. The intrinsic fluorophores of proteins are the aromatic amino acids which render the protein its intrinsic fluorescence. ALA has four Trp at four different sites (Trp-118, Trp-60, Trp-104, and Trp 26), and these Trps are major contributors to the intrinsic fluorescence.12 Therefore, it is possible to use the intrinsic Trp fluorescence quenching in ALA as a tool to study the interactions between CA and ALA. It was reported that the intrinsic fluorescence of ALA is very sensitive to change in its microenvironment.30 In Figure 1A, it is seen that ALA at pH 7.4 at 288 K gave a maximum fluorescence peak of around 329 nm because ALA is present in the holo form; if the calcium was removed from ALA, the fluorescence intensity increased and the wavelength maximum also shifted toward a higher value (∼336–340 nm).19 In our case, we have used holo ALA which gives maximum fluorescence around 329 nm, but the fluorescence intensity decreased, and the peak position was unchanged with respect to increase in temperature. The quenching phenomenon caused by solvent-exposed collisional quenching is responsible for the decrease in fluorescence intensity with increasing temperature.31Figure 1B–D demonstrates the effect of CA on the fluorescence emission spectra of ALA at three different temperatures. As shown in the figures, the intrinsic fluorescence intensity value of ALA at 329 nm decreased with increasing CA concentration. The decrease in fluorescence intensity observed after adding CA was due to molecular quenching. The fluorescence quenching data designated that the ALA conformation changed and that an intermolecular energy transfer occurred between CA and ALA. These quenching results indicate that CA could bind with ALA through the quenching mechanism. Similarly, we have observed the effect of ethanol on the fluorescence emission of ALA, and we used 1% (V/V) at pH 7.5 and 298 K, as can be seen in Figure S1 in the supplementary file. On excitation at 280 nm, ALA gives an emission maximum of 329 nm with relative fluorescence intensity (RFI) at 395. With 1% (V/V) ethanol addition, no change was observed in the emission spectrum which confirms that the tertiary structure of ALA is unaltered in the presence of 1% (V/V) ethanol.

Figure 1.

Figure 1

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(A) Fluorescence spectra of ALA in the absence of CA at 288 (black trace), 298 (red trace), and 310 K (blue trace). ALA solution fluorescence spectra without and with different concentrations of CA 288 (B), 298 (C), and 310 K (D). All of the samples have an ALA concentration of 2.0 μM.

Fluorescence Quenching Mechanism and Binding Constant

Fluorescence quenching primarily occurs through two different mechanisms: dynamic quenching and static quenching.32 The fluorescence quenching mechanisms can be determined by observing the changes in KSV values at different temperatures. In dynamic quenching, the value of KSV increases with the increase in temperature because of faster diffusion and larger amounts of collisional quenching. However, in static quenching, the KSV values decrease with the increase in temperature.33 The fluorescence spectral data were examined using the Stern–Volmer equation to calculate the quenching constant Ksv and the bimolecular rate constant (Kq) defined in eq S1 to elucidate the quenching mechanism between the CA and ALA systems. In this study, we are interested to find out which quenching mechanism plays the leading role. To assess it, the fluorescence intensities of the CA-ALA system were analyzed by plotting F0/F versus [Q] at three different temperatures (288, 298, and 310 K) as shown in Figure 2A, and the calculated KSV values are provided in Table 1. The three temperatures have been chosen by keeping an eye on the best residual activity and thus the stable native state of ALA, as reported by several studies. In Figure 2A, we can see that the regression lines under every temperature reveal a good linear relationship, and their slope (KSV) decreases from 3.4 to 3.2 and to 2.9 (× 104 L mol–1) at 288, 298 and 310 K. The decrease in KSV values with the increase in temperature suggests that the nature of quenching taking place in between CA and ALA is static type. As already reported, if the Ksv value decreases with an increase in temperature, the quenching mechanism will be static type.34 The modified Stern–Volmer plot, log[(F0/F) – 1] versus log [CA], is plotted in Figure 2B. The slope and intercept of the modified Stern–Volmer plots were used to calculate the number of binding sites (n) and the binding constant (Kb) using eq S3. The value of Kq was found to be 1000 times greater than the maximum scatter collision quenching constant (2.0 × 1010 mol–1 L s–1) of other biomolecules, indicating that the quenching is not dynamic, but rather CA binds with ALA and forms a strong complex.35 The overall results, particularly the value of Ksv, suggest that the interaction is static. The values of Kb and n for CA were calculated at various temperatures, and the results are shown in Table 1. The data show that a decrease in Ksv or a decrease in Kb with increasing temperature clearly indicates static quenching.36 With the increase in temperature, the ALA seems not to have major conformational alteration, except for some minor rearrangements, in the temperature range of 288–310 K. Here, CA shows a strong binding with ALA with a binding constant of more than 10–4. Recently, ALA and fatty acid binding studies are popular due to the advent of HAMLET which is an ALA-OA complex. HAMLET is toxic to malignant cells but not to the differentiating healthy cells. The bovine counterpart BAMLET also shows similar activity.20

Figure 2.

Figure 2

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Stern–Volmer plots for CA (A) as well as the log[(F0/F) – 1] vs log[CA] plots (B) for the binding of CA with ALA at the three different temperatures.

Table 1. Binding Parameters of ALA with CA at 288, 298, and 310 K are Shown in below Table, and these Parameters Were Calculated from Fluorescence Quenching Data.

s. no. T (K) Ksv (× 104 M–1) Kq (× 1013 M–1 s–1) R2 n Kb (× 104 M–1)
1. 288 3.4 3.4 0.997 1.0 5.0
2. 298 3.2 3.2 0.997 1.0 3.1
3. 310 2.9 2.9 0.997 0.95 1.5
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Thermodynamics of ALA-CA Interaction

Small molecules mostly interacted with proteins via four binding forces: electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals attractions. The thermodynamic parameters, particularly the enthalpy change (ΔH) and the entropy change (ΔS), are critical for determining which binding forces work in the reactions.36 The Van’t Hoff equation can be used to calculate these parameters (eqs S4 and S5). We have already discussed the binding constant, estimated at three different temperatures, while assuming that ALA did not undergo structural degradation at any of these temperatures. The thermodynamic parameters were calculated from the linear second law of the thermodynamics plot (Figure 3) based on the binding constants of CA to ALA at three different temperatures, and the calculated values are tabulated in Table 2. The obtained enthalpy-entropy changes are primarily caused by the interaction of CA molecules with ALA. As shown in Table 2, ΔG is negative in all conditions, indicating that the binding process is spontaneous. The ΔH and ΔS values for the complex formation between CA and ALA are −39.06 and −44.89 KJ/mol, respectively. As a result, this type of CA-ALA interaction is an exothermic reaction with a negative ΔS value. CA stabilizes ALA, and the negative ΔS value indicates that the binding solvent to the protein molecule in or near the binding pockets was not disturbed. The signs and magnitudes of ΔH and ΔS can also be used to determine the interacting forces between a protein and a ligand.37 Our findings show that the thermodynamic values of ΔH and ΔS are negative, implying that hydrogen bonding is the dominant force between CA and ALA. It echoes previous findings in which nintedanib interacts with BSA via hydrogen bonds, as confirmed by fluorescence results.38

Figure 3.

Figure 3

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van’t Hoff plot shows the temperature dependence of Kb. ALA fluorescence quenching by CA at 288, 298, and 310 K.

Table 2. After Binding of CA with ALA at 288, 298, and 310 K, the Thermodynamic Changes, Specifically the Enthalpy Change (ΔH), Entropy Change (ΔS), and Gibbs’ Free Energy Change (ΔG), Are Presented.

s. no. T (K) ΔG (KJ mol–1) ΔH (KJ mol–1) ΔS (cal Mol–1 K–1)
1 288 –25.90 –39.0687 –44.8956
2 298 –25.66
3 310 –24.91
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Synchronous Fluorescence Spectroscopy

The fluorescence quenching was measured using synchronous fluorescence, which provides information about the protein’s conformational changes. Excitation at Δλ = 60 and 15 nm, respectively, was used to analyze the molecular environment in the vicinity of choromophoric residues, that is, tryptophan and tyrosine residues in the protein.39 The synchronous fluorescence spectrum of ALA exhibits the microenvironment change around tryptophan and tyrosine residues (Figure 4A,B). From the figure, it can be noted that the fluorescence intensity was continuously decreasing due to the continuous increase of CA concentration. The decrease in synchronous fluorescence may be due to more conformational changes around tryptophan and tyrosine residues. Interestingly, no shift in wavelength maximum was found around both the chromophores. The quenching in synchronous fluorescence indicates that the tryptophan and tyrosine residues are in a more hydrophobic environment, and the quenching level is almost the same around both residues.

Figure 4.

Figure 4

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Synchronous fluorescence spectra of ALA in the presence of different concentrations of CA at 288 K: Δλ = 60 nm. (A) and Δλ = 15 nm (B). The ALA concentration is fixed at 2.0 μM in all samples, while the CA concentration is varied from 0.0 to 9.0 μM.

UV–vis Absorption Spectroscopy

UV–vis absorption spectroscopy is one of the most useful techniques for studying ligand-induced conformational changes in proteins.40Figure 5 depicts the UV–vis absorption spectra of ALA in the absence and presence of different concentrations of CA. In Figure 5, ALA without CA had an absorption peak around 280 nm (A280), which is credited to the absorption of tryptophan, tyrosine, and phenylalanine residues of ALA.41 However, with a gradual increase of CA, the peak intensity of ALA at 280 nm increases with a blueshift in the wavelength maximum, which designates that the interaction between CA and ALA takes place and the aromatic residues of ALA are exposed toward polar environments. The change in peak intensity is dependent on CA concentrations. The UV–vis results suggest that the increase in absorbance (A280) in response to the increase in CA is due to the complex formation between ALA and CA.

Figure 5.

Figure 5

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UV–vis spectra of ALA at pH 7.4 in the absence (black) and presence of 30 (red), 45 (green), and 60 μM CA (blue). The absorbance at 280 nm (A280) is monitored for changes in absorbance as the concentration of the substance increases (CA). The ALA concentration in all samples was 15.0 μM.

CD Spectroscopy

Far-UV CD is a very good spectroscopic technique used in biology and chemistry to determine the changes in the protein secondary structure.42 Spectra in the far UV range of ∼200–250 nm provide detailed information on protein polypeptide backbone conformations. Intermolecular forces that are responsible for holding secondary and tertiary protein structures are observed during ligand interactions with protein molecules, which further aid in protein conformational changes. The far-UV CD spectra of ALA in the absence and presence of CA were recorded to characterize the changes in the secondary structure, as shown in Figure 6. The ALA CD spectrum shows two negative minima at 208 and 222 nm, which are characteristic of the ALA’s helix structure. The ALA far-UV CD spectrum signature matches other published reports.43 The ellipticity of the far-UV CD spectrum of ALA increased after interaction with CA, and the shape of minimum 208 and 222 nm remained the same except for a slight increase in the ellipticity. The increase in negative ellipticity clearly suggests that the ALA gained more α-helical structure after interacting with CA. The secondary structure of ALA is slightly modified due to binding with CA, and the modification is shown to stabilize the α-helicity of ALA molecules. Other reports also suggest similar secondary structural changes when HSA interacted with lactic acid enantiomers.44

Figure 6.

Figure 6

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Far UV-CD spectra of ALA (15.0 μM) without CA (black) and with CA. The CA concentrations were (1.0 (red), 4.0 (green), and 7.0 (blue) μM at pH 7.4.

Detection of Exposure of Hydrophobic Clusters by ANS Binding

ANS is considered a gold standard to evaluate the hydrophobicity on the accessible surface area (ASA) of a protein. Free ANS, which absorbs at 380 nm and shows maximum emission at 480 nm, shifts toward a higher wavelength once bound to the hydrophobic patches of a protein ASA. We have used ANS binding to evaluate

  • 1.

    whether the presence of 1%(V/V) ethanol alters the ANS binding toward ALA?

  • 2.

    whether 30 μM CA binding will alter the ANS binding on ALA?

Figure 7 shows the ANS fluorescence in the presence of 3.0 μM ALA, in the presence of 3.0 μM ALA and 1% (V/V) ethanol, and in the presence of 3.0 μM ALA, 1% (V/V) ethanol, and 30 μM CA. The results show that the presence of 1% (V/V) ethanol will not alter the ANS binding on ALA. Ethanol is a known disrupter of water bonding. However, 1% (V/V) ethanol concentration is too less to make any observable change in the ANS and ALA interaction. Further, no change has been observed in the ANS and ALA complex fluorescence in the presence of 30 μM CA under the same circumstance. It indicates that the binding of CA with ALA played little role in the alteration of the surface hydrophobicity of ALA.

Figure 7.

Figure 7

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ANS fluorescence of samples. (i) ALA with 60 mM ANS (black), (ii) ALA with 1% EtOH+ 60 mM ANS (red), and (iii) ALA with 9.0 μM CA + 1% EtOH +60 mM ANS (blue).

Molecular Docking of CA on ALA

Computational docking is widely used to study protein–ligand interactions, particularly in drug discovery and development.45 As shown in Figure 8, CA is docked within the ALA binding site with a minimum binding energy of −4.7 kcal/m. As a result, CA becomes stable in the protein’s active site, as shown in Figure 8A. The amino acid interaction is shown in Figure 8B. Among the interactions, we observe three hydrogen bonding interactions with Ser112 and Lys108, providing the complex’s stable conformation.

Figure 8.

Figure 8

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CA binding with ALA. (A) Molecular surface of the protein ALA is shown in green, and the ligand CA (red) is shown in the binding pocket. (B) Amino acid interactions in the binding pocket of ALA.

MD Simulation of ALA and CA Complex

MD simulation is a powerful method to understand the dynamic nature of protein structures at an atomic level.46 The conformational changes of ALA in complex with CA were assessed using RMSD. The RMSD represents the stability and structural convergence of the protein during the simulation, and we determined the RMSD calculation after interacting with the ligand molecule, as shown in Figure 9. From the RMSD, it was observed that the protein–ligand complex structure was flexible during the initial simulation period from 0.4 to 1.6 nm (between 0 and 32 ns). The trajectory from 32 to 75 ns showed low fluctuations. After 75 ns, the complex acquired equilibrium up to the final simulation with minimal changes of 0.25 nm and closed at 1.10 nm. Finally, the complex trajectory was equilibrated and maintained its stability with minute fluctuations from 0.06 to 100 ns, closing at 1.35 nm, more significant in the protein–ligand complex.

Figure 9.

Figure 9

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Backbone RMSD analysis of the 100 ns MD simulation trajectories of a protein–ligand complex.

Different interactions, such as hydrogen bonds, electrostatic bonds, hydrophobic bonds, and so forth, stabilize the protein–ligand complexes. Hydrogen bonds are crucial interactions that play a vital role in stabilizing the receptor-ligand complex. As a result, we calculated the number of hydrogen bonds in the ALA and CA complexes as a function of time during the 100 ns MD simulation. The results revealed a maximum of three hydrogen bonds in the complex, out of which two hydrogen bonds were observed throughout the MD simulation, as shown in Figure 10. The observed hydrogen bonding reflects the ligand’s effective binding to the complex (i.e., ALA and CA complex).

Figure 10.

Figure 10

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Hydrogen bond interaction between ALA and CA.

Conclusions

In this work, we examined the interaction between CA and ALA using several spectroscopic and computational methods. These data show that complexes formed between CA and ALA as a result of interactions. The quenching results indicate that CA binds to ALA in a static quenching manner. The thermodynamic data revealed that the binding process is spontaneous and exothermic. The fluorescence data at three different temperatures suggested that the CA interacted with ALA molecules mainly through hydrogen bonding. The UV–vis and far-UV CD results indicate that the tertiary and secondary structure conformations of ALA are changed due to interaction with CA. CA is the most valuable ingredient in the food industry, so our results are important in the applications of CA in the food industry and drug formulation.

Acknowledgments

The authors are grateful to the Researchers Supporting Project Number (RSP2023R360), King Saud University, Riyadh, Saudi Arabia.

Glossary

Abbreviations

caffeic Acid

CA

α-lactalbumin

ALA

8-anilino-1-naphthalenesulfonic acid

ANS

molecular dynamic

MD

circular dichroism

CD

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c01755.

  • Fluorescence quenching data analysis methods; methods of inner filter effects calculation;and intrinsic fluorescence measurements (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao3c01755_si_001.pdf (156.7KB, pdf)

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