Purification, Characterization and Spectrofluorometric Study of Buffalo Lactoferrin Coupled to Amoxicillin

Abstract

Antibiotic resistance raises concerns about their residues in food and the environment. To produce antibiotic-free dairy products, countries explore new strategies to reduce antibiotic doses in mastitis treatment. Lactoferrins (LF), iron-binding glycoproteins, exhibit species-specific glycosylation affecting their properties. While formulations of bovine lactoferrin have been tested with antibiotics showed synergistic properties increasing the antibacterial properties, other interactions of lactoferrin isoforms with antibiotics remain unclear. This study isolates, purifies, and characterizes buffalo lactoferrin (Bf-LF) and its interaction with amoxicillin. Bf-LF was purified using a Sephacryl S-100 column and monitored spectrofluorometrically. Spectroscopic analysis of Bf-LF interaction with amoxicillin reveals that increasing amoxicillin concentration reduces Bf LF fluorescence, indicating conformational changes. The Stern-Volmer quenching constant (K_SV) and binding constant (K_a) measured at 298 K shows values around (2.79 ± 0.29) × 10⁴ L mol^-1 and (0.300 ± 0.04) × 10⁴ L mol^-1, respectively. UV-Vis spectroscopy shows a red shift (258.08 to 271.83 nm) implying Bf-LF-amoxicillin binding. In silico studies suggest polar interactions between amoxicillin and specific Bf-LF amino acids.

Keywords:
whey protein; antibiotics; spectrofluorometry; UV-Vis spectrophotometry; protein drug; molecular docking

Introduction

Lactoferrin (LF) is an iron-binding glycoprotein, member of the transferrin protein family, isolated from secretions of mammalian species with a molecular weight of around 80 kDa.¹-³ LF is present in colostrum, milk and in most mucosal secretions, including uterine fluid, tears, saliva, and nose secretions of numerous mammalian species, such as cows, humans, goats, horses, dogs, and various rodents.⁴ Bovine LF has a variety of biological functions, such as immunomodulatory properties, antimicrobial capacity against bacteria, parasites, fungi and viruses, and antioxidant, anti-inflammatory and anticancer activity.⁵,⁶

LF is composed of a polypeptide chain, that consists of two symmetric globular lobes (N-lobe and C-lobe) connected by a short α-helix.¹,³ In each lobe, two domains, referred to as N1 and N2, or C1 and C2, enclose a deep cleft containing the amino acid residues for the iron-binding site. The iron binding mechanism remains conserved for both human and bovine LFs. Four different amino acid residues (aspartic acid-Asp 60, tyrosine-Tyr92, Tyr192, and histidine-His253) are involved in binding the Fe³⁺ ion.¹,⁷ According to Ostertag et al.,⁸ lactoferrin binding constants (K_D) show values in a range of 10²⁰ to 10²⁴ M^-1, indicating the high affinity for Fe³⁺. The LF molecule can bind two iron ions reversibly together to two synergistically bound carbonate ions (CO₃^2-).⁹

Due to genetic polymorphism, LF undergoes several pos-translational modifications such as glycosylation, impacting important roles in the structural conformation and bioactivity of proteins. Although glycosylation is observed on the molecules of all LFs, the number of glycosylation sites is species related, resulting in variation in their molecular stability and resistance to proteolysis.⁴,¹⁰

The biological functions of LF are related to its iron binding ability. Studies⁴,¹¹ have shown that binding of iron to the LF molecule improves the stability of its structural conformation. A previous research¹² has demonstrated a synergetic effect where the combination of LF with penicillin increased the inhibitory effect of penicillin on the growth of Staphylococcus aureus isolated from bovine by two to fourfold. Subsequently, LF has been incorporated into new strategies to combat microbial infections caused by drug resistant bacteria. In those studies, LF is employed as an adjunct to beta-lactam antibiotics.¹³,¹⁴

Mastitis is an inflammatory disease in the mammary glands resulting from microbial infections,¹⁵-¹⁷ by pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, Mycoplasma spp., Enterobacter aerogenes, Klebsiella pneumoniae, Escherichia coli, and Corynebacterium sp.¹⁵,¹⁸ It is a sanitary and economic problem for the dairy industry, resulting in reduced production, poor milk quality and high treatment costs, while impacting the welfare and longevity of the animal stock.¹⁷,¹⁹ Although new alternatives are emerging for the treatment of mastitis, administration of antibiotics remains the main strategy.¹⁵ However, the emergence of antibiotic resistance and the accumulation of antibiotic residues in the animal-derived products have raised concerns within global public health systems regarding these therapies.¹⁷,²⁰ Due to their toxicity, antibiotic residues produce adverse and harmful effects on consumer health such as resistance to antibiotics, allergic reactions, and disruption of the digestive system.¹⁶,¹⁸ Multiple European countries, including the Netherlands, Italy, Finland, Germany, and Spain have created legislation and databases for monitoring the use of antibiotics and their residues in animal products.²¹

Although mastitis is a severe disease impacting both bovines and non-bovines, e.g., Murrah and Mediterranean buffaloes,²²,²³ most of the studies on new therapies, including animal-derived antimicrobials, have been performed on bovines.²³,²⁴ Recently, molecular studies have identified variations in the protein-coding gene (serine/threonine kinase 3-AKT3) involved in the inflammatory response against mastitis in cow and buffalo.²⁵

A search in the RCSB Protein Data Bank (RCSB PDB) produced relevant studies on the 3D structure of native and recombinant bovine and human LF complexed with drugs. These studies provide very interesting insights into the dynamics of proteins. For instance, crystallization studies reveal differences in the conformation of apo-LF from human, bovine, camel, and caprine origin in the region of the N- and C-lobes influencing the iron binding capabilities of both lobes; this could potentially alter their physiological properties.²⁶-³²

Spectroscopy analyses such as UV spectrophotometry and fluorometry are common techniques used in the characterization of protein-drug interaction.³³ These spectroscopic techniques are very useful to identify differences in the interaction mechanism of LF with drugs, showing changes in the protein conformation and information about the stability of the complex formed. In the literature one can find spectroscopic studies of the interactions of bovine LF and whey protein with drugs such as tetracycline hydrochloride,³⁴ sulfonamides,³⁵ 3,3’-diindolylmethane (DIM),³⁶ flavonoids quercetin, luteolin and naringenin,³⁷ sugar alcohol compounds with xylitol,³⁸ and polyphenols theaflavins.³⁹

Because of the multifunctional properties of LF, the aim of this study was to isolate, purify, biochemically characterize, and analyze the interaction of buffalo lactoferrin (Bf-LF), a less studied LF, with amoxicillin, a beta-lactam antibiotic routinely used for mastitis treatment in bovines.⁴⁰

Experimental

The study was carried out at the Federal University of Alagoas, situated in the North-East of Brazil. The milk samples from mixed-race buffaloes with Murrar breed predominance came from the Castanha Grande dairy farm, in São Luiz do Quitunde also in Alagoas.

Sodium hydroxide, hydrochloric acid, Sephacryl S-100, ammonium sulfate, acrylamide, β-mercaptoethanol, tris(hydroxymethyl) aminomethane, glycine, monohydrate potassium phosphate, dihydrotic potassium phosphate, molecular weight protein markers, acrylamide, ammonium persulfate, amoxicillin, LF from bovine milk ≥ 85% purity by sodium dodecyl sulfate (SDS) electrophoresis (L9507, Sigma-Aldrich), N,N,N’,N’ tetramethyl ethylene diamine (TEMED), sodium dodecyl sulfate (SDS), Coomassie Brilliant Blue G-250, Coomassie Brilliant Blue R-250, phosphoric acid, glycerin, agarose, quercetin were all obtained from Sigma-Aldrich (St. Louis, MO, USA).

Milk processing

After milking, samples of buffalo milk were stored at 4 °C to avoid protein denaturation. The milk samples were processed according to Vijayan et al.,⁴¹ and Mahala et al.,⁶ with some modifications. Milk fat removal was performed by centrifugation (2000 ×g, 30 min, 4 °C), after which the skimmed milk was acidified to pH 4.6 (isoelectric point of casein) with 1 mol L^-1 HCl at 20 °C. Samples of the acidified whey were used in the further procedures.

Lactoferrin purification

Upon achieving pH 6.8 through neutralization, whey samples were submitted to saline precipitation (0-20, 20-40, 40-60, and 60-80%) with ammonium sulfate (m/v) and all fractions were centrifuged at 10000 ×g for 30 min at 4 °C. The pellets were re-suspended in buffer and further dialyzed overnight against a 0.2 mol L^-1 potassium phosphate buffer at pH 7.7. All dialyzed samples were analyzed fluorometrically, scanning for Bf-LF from 300 to 550 nm on excitation and emission wavelengths of 295 nm.³³

An aliquot of (0.3 mL) of the dialyzed saline fraction containing higher protein content and showing a characteristic LF fluorometric spectrum (1.713 mg mL ¹) was applied to a gel filtration (Sephacryl S-100 HR, 1 cm × 60 cm) connected to a chromatography system AKTA Pure M1 (EG) (Maidstone, Kent, United Kingdom), with flow rate adjusted to 0.1 mL min^-1; the column was equilibrated and eluted with a 0.1 mol L^-1 potassium phosphate buffer (pH 7.7). All eluted fractions were analyzed by the fluorometer, and protein content was measured.

Fluorescence measurement

LF in the samples was monitored fluorometrically (fluorescence spectrophotometer Shimadzu, model RF 5301pc) (Kyoto, Japan) during every step of the purification protocol. For that purpose, aliquots of 100 µL were added to a 1.0 cm quartz cell with 2.5 mL of a 0.05 mol L^-1 Tris-HCl buffer, pH 7.0, containing 0.01 mol L^-1 NaCl. Under the apparatus condition of both entrance and exit slit width being 5 nm and a scanning speed of 240 nm min^-1, a fluorescence and a fluorescence quenching spectra were acquired. Fluorescence quenching spectra were obtained at excitation and emission wavelengths of 295 and 300-550 nm, respectively, according to the Guo et al.³³ Fluorescence spectra of commercial bovine LF (BLf-Sigma) (L9507) (Sigma-Aldrich, St. Louis, MO, USA) were also acquired as standard.

Protein quantification

Protein concentration of the samples was measured according to Bradford,⁴² using Coomassie brilliant blue G-250 dye and bovine serum albumin as the standard. A diluted aliquot (100 μL) was added to 2.5 mL Bradford reagent, and the absorbance was read at 595 nm. The assays were conducted in triplicate.

Electrophorese (SDS-PAGE)

The SDS-PAGE method was performed according to Laemmli.⁴³ PAGE under denaturing conditions (SDS PAGE) was executed using 8% acrylamide gel containing 0.5% TEMED and run in an ice bath at 90 V. Gels were stained with a Coomassie Brilliant Blue G-250 solution at room temperature while gentle shaking until colored protein bands developed. Commercial bovine LF (BLf-Sigma) (L9507), (Sigma-Aldrich, St. Louis, MO, USA) was used as LF standard.

Fluorometric and UV-Vis assays of the coupled lactoferrin preparations

The interaction of Bf-LF and amoxicillin was analyzed fluorometrically (fluorescence spectrophotometer Shimadzu, model RF-5301pc) (Kyoto, Japan) and by UV-Vis absorption. Fluorescence emission spectra were measured from 200 to 500 nm at an excitation wavelength of 280 nm.³⁴

The absorbance spectra were recorded by a UV Vis spectrophotometer (PerkinElmer, model Lambda 2) (Waltham, Massachusetts, USA) in a range of 200 to 500 nm.³⁷ The reaction mixture (2.5 mL) contained 0.05 M Tris-HCl buffer (pH 7.0, 0.01 mol L^-1 NaCl), 100 µL of Bf-LF preparation (0.3 mg mL^-1 protein), and amoxicillin (stock solution of 5.0 × 10^-5 mL L^-1). Nine amoxicillin concentrations (0, 1, 2, 3, 4, 5, 6, 7, and 8 × 10^-6 mol L^-1) were added to the reaction mixture.

The Stern-Volmer quenching constant (K_SV) and binding constant (K_a) were measured at 298 K according to Shang et al.⁴⁴

Molecular docking

The molecular docking between the Bf-LF model and amoxicillin model was conducted for predicting the binding modes for this drug. The 3D-structure of buffalo lactoferrin (PDB ID: 1CE2) was obtained from the Protein Data Bank (RCSB PDB) website,⁴⁵ having 2.5 Å resolution by X-ray diffraction crystallography.⁴⁴ The 3D-structure of amoxicillin was downloaded from the NCBI database of PubChem (Bethesda, MD, USA),⁴⁶ which posteriorly was energetically minimized by using the ArgusLab software, v. 4.0.1,⁴⁷,⁴⁸ by employing the Parameterized Method 3 (PM3). All co-crystallized ligands, water molecules, and ions were removed by preprocessing the Bf LF structure on AutoDock Tools, v. 1.5.7.⁴⁹,⁵⁰ Posteriorly, the cocrystallized ligand was used in the redocking step for validating our protocol, generating a binding pose with an RMSD value of 0.85. Then, the AutoDock Vina software,⁵¹,⁵² via Python Prescription (PyRx-Virtual Screening Tool) v. 0.8 interface,⁵³,⁵⁴ was used to perform all docking simulations involving amoxicillin. Furthermore, all intermolecular interactions (H-bond, hydrophobic, and van der Waals) were individually analyzed using AutoDock Tools v. 1.5.7 (TSRI, San Diego, USA).⁵⁰,⁵⁵ Finally, all illustrations and RMSD values were generated by using PyMol^® software, v. 3.0.2.⁵⁶,⁵⁷

Statistical analyses

The results were expressed as mean values ± standard deviation, and mean values were compared using two-way analysis of variance (ANOVA).

Results and Discussion

Processing of the milk samples

Table 1 and Figure 1 show the ammonium sulfate fractionation of the buffalo neutralized whey and their fluorescence spectra, respectively. The precipitates from 20 40 and 40-60% saturations with 0.8 and 4.5 mg protein mL^-1 (Table 1) yielded spectra with fluorometric intensity and an emission peak at 332 nm (Figure 1). Both precipitates exhibit a fluorometric intensity spectrum similar to the standard bovine LF spectrum under the same excitation 295 nm and emission 300 550 nm wavelength conditions (Figure 1). These results show that Bf-LF was precipitated at these saturation levels. Since 40 60% saturation contains a higher protein content (Table 1), this precipitate was destined for further purification.

Table 1
Ammonium sulfate fractionation of the buffalo neutralized whey

Fractions	Volume / mL	Protein / (mg mL-1)	Total protein / g
Neutralized whey	350	3.2 ± 0.40	1.1 ± 0.14
0-20% supernatant	330	3.4 ± 0.17	1.1 ± 0.06
20-40% supernatant	310	3.2 ± 0.25	0.9 ± 0.08
20-40% resuspend precipitated	3.75	0.8 ± 0.02	0.003 ± 0.00
40-60% supernatant	280	1.7 ± 0.062	0.5 ± 0.02
40-60% resuspend precipitated	2.92	4.5 ± 0.14	0.01 ± 0.00
60-80% supernatanta	260
60-80% resuspend precipitateda	11.08

• ^a
  Even after two overnight dialysis, the high salt concentration in these samples interfered in the Bradford method measurements to quantify their protein content. Values are mean plus or minus (±) one standard deviation.

Figure 1
(A) Fluorescence spectra of salt precipitation fractions (salting out) of buffalo whey in excess of ammonium sulfate. (B) Fluorescence spectra of buffalo whey (0.842 mg mL^-1) and bovine LF (Sigma-Aldrich, L9507) (0.842 mg mL^-1).

The fluorescence spectrum of Bf-LF resembles the fluorescence spectrum of the standard LF (Figure 1). This indicates that spectrofluorometry can be used as a tool to monitor the isolation of LF. Furthermore, Chen et al.,⁵⁸ and Huang et al.,³⁷ report fluorometric intensity spectra of bovine LF at 332 and 334 nm, respectively, with wavelength conditions of excitation in 290 nm and emission in 300 550 nm.

Purification of buffalo lactoferrin

The further purification of the 40-60% resuspended precipitate was carried out using size exclusion chromatography on a Sephacryl S-100 column as described in the Experimental section. The chromatographic profile of the elution shows three significant peaks within the 12 16, 17-22, and 23-26 fractions (Figure 2). Based on the fluorometric spectra of these peaks, peak 1 (fraction 12 16) exhibits a spectrum comparable to the spectrum of bovine LF (Figure 3). Subsequently, the peak 1 volume was reduced by a concentrator to a protein concentration value of 0.102 mg mL^-1.

Figure 2
Chromatographic profile of the purification of LF from buffalo milk in size-exclusion Sephacryl S-100 column.

Figure 3
(A) Fluorescence spectra of eluted fractions from the size-exclusion Sephacryl S-100 column (pH 7.7; 290 nm). A pool of fractions was made for each peak: peak 1 (fractions 12-16), peak 2 (fractions 17-22), peak 3 (fractions 23-26), peak 4 (fractions 27-29) and peak 5 (fractions 30-35). (B) Fluorescence spectra of buffalo whey (0.842 mg mL^-1) and bovine LF (Sigma-Aldrich, L9507) (0.842 mg mL^-1).

Bf-LF was partially purified in the Sephacryl S-100 molecular exclusion chromatography column. The fluorometric spectrum of the first peak (fraction 12-16 in Figure 2) is similar to the bovine LF spectrum (Figure 3). Narmuratova et al.⁵⁹ purified mare LF of Kazakhstan in a Sephadex G-100 column where the elution profile showed two peaks with the second peak rich in LF. Abbas et al.⁶⁰ used two subsequent chromatography steps using a cation exchanger (CM-Sephadex C-50) and gel filtration chromatography (Sephadex G-200) to get a single peak rich in LF. Camel LF and bovine LF were also partially purified using cation exchange chromatography in a SP-Sepharose size-exchange Sephacryl S-300.⁶¹

Polyacrylamide gel electrophoresis (PAGE)

In Figure 4, SDS-PAGE fingerprints obtained from peak 1 (fraction 12-16) of the Sephacryl S-100 column elution (lanes 1 and 1’) are shown. Both lanes display a band around 87 kDa, which is comparable to the bovine LF from Sigma-Aldrich used as standard (M_BLF).

Figure 4
SDS-polyacrylamide gel electrophoresis (12%) of the purified Bf-LF stained with Coomassie Brilliant Blue (CBB-G250). Lane M_BLF: bovine LF (Sigma-Aldrich, L9507, 87 kDa (15.10 μg) as standard, lane 1: pooled fractions 12-16 (14.40 μg), lane 2: buffalo neutralized whey (5.10 μg), lane 1’: pooled fractions 12-16 (15.10 μg).

In this study, Bf-LF from milk whey of mixed-race buffaloes, with Murrar breed predominance, SDS-PAGE of the 12-16 fraction (peak 1) showed a band of around 87 kDa, which is comparable to bovine LF from Sigma (Figure 4). Several studies have reported successful purification of LF from different sources, showing variation in their molecular weight (Table 2). These variations in the molecular weight of LFs isolate from mammals may be species related and due to the carbohydrate content in their molecules.⁵⁹

Table 2
Molecular weight among LFs

Species	Start material	Molecular weight / kDa	Reference
Buffalo (mixed-race with Murrar breed)	whey	ca. 87	present work
Buffalo	colostrum	80	62
Buffalo	recombinant expression in yeast systems	80	63
Bovine	whey	70	64
Bovine	whey obtained from fresh cottage cheese	80	65
Bovine	whey obtained from cheese production	80	66
Bovine	milk	87	commercial bovine LF (L9507) Sigma-Aldrich
Camel	milk	ca. 77	6
Goat	colostrum	80	60
Goat	colostrum	82	67
Goat	milk	79	68
Goat (Malabari)	colostrum	80	41

Effects of amoxicillin on Bf-LF fluorescence emission spectra

The fluorometric intensity of Bf-LF peaked at approximately 337 nm and decreased continuously upon amoxicillin titration (Figures 5A and 5B),⁴⁴ showing an interaction between the antibiotic and Bf-LF. In addition, a hypochromic shift from 337.02 to 335.32 nm in the maximum wavelength of Bf-LF was observed. This hypochromic shift may be due to a reduction in polarity accompanied by an increase in the hydrophobic interaction promoted by typtophan-Trp. Figures 5C and 5D show the Stern-Volmer equation of fluorescence quenching of Bf LF due to its interaction with amoxicillin and the binding constant K_a respectively (K_SV = 2.493 × 10⁴ L mol ¹, K_a = 0.302 × 10⁴ L mol^-1). Binding studies of bovine and goat lactoferrin with small molecules shows values for the binding constant with similar magnitude values (10⁴ L mol ¹),³⁶ Sun et al.,³⁴ Huang et al.,³⁷ and Ma et al.⁶⁹ The binding constant obtained for the Bf-LF-amoxicillin coupling indicates a good interaction.

Figure 5
(A) The fluorescence quenching spectra of Bf-LF in the presence of different concentrations of amoxicillin C_amoxicillin= 10^-6 mol L^-1; from (a) to (i): 0, 1, 2, 3, 4, 5, 6, 7, 8. (B) Fluorescence spectra of amoxicillin (2.5 mol L^-1), Bf-LF (0.842 mg mL^-1) and Bf-LF + amoxicillin (3 × 10^-6 mol L^-1) (pH 7.7; 290 nm). (C) Stern-Volmer and (D) binding constant and number of binding sites of the interaction of amoxicillin with Bf-LF.

Figure 6A shows UV-Vis spectra of the influence of amoxicillin titration on Bf-LF. Two absorptions peaks were observed for amoxicillin at wavelength 228 and 270 nm (Figure 6B). Bf-LF showed a maximum absorption peak at 270.5 nm. Under amoxicillin titration Bf-LF exhibited a red shift from 270.5 to 280.7 nm, a red shift of 10.2 nm (Figure 6C). The absorption peak heightened with the rise in amoxicillin concentration, indicating binding of amoxicillin to Bf-LF. According to concept, isosbestic point is the wavelength in which the absorbance of two or more species are the same. Based on that, the isobestic point of buffalo lactoferrin-amoxicillin complex formation is around 280-300 nm interval. Additionally, the decrease in the intensity of absorption peak at 228 nm of amoxillin in the complex (Figure 6B, inset), corroborates with the interaction. A comparison between the spectra of both is shown in Figure 6D.

Figure 6
(A) UV-Vis spectra of Bf-LF in the presence of amoxicillin. C_amoxicillin = 10^-6 mol L^-1; from (d) to (k): (0, 1, 2, 3, 4, 5, 6, 7, 8) × 10^-6 mol L^-1. (B) UV Vis spectra of amoxicillin (2.5 × 10^-6 mol L^-1), Bf-LF (0.3 mg mL^-1) and Bf-LF + amoxicillin (8 × 10^-6 mol L^-1). (C) Red shift of UV-Vis spectra of Bf-LF and amoxicillin. (D) Comparison of absorption peak of the UV-Vis spectra of Bf-LF (0.3 mg mL^-1) with the spectra of amoxicillin (2.5 × 10^-6 mol L^-1) and Bf-LF + amoxicillin (8 × 10^-6 mol L^-1).

The amoxicillin concentration also caused distinct changes in the UV-Vis absorption spectrum of Bf-LF. This change in the UV-Vis spectrum reinforces the hypothesis of biochemical interaction of the Bf-LF and amoxicillin. Sun et al.³⁴ obtained a similar result when studying the interaction between bovine LF and tetracycline hydrochloride. The environment around the amino acid residues is determined by the conformation of the protein molecule. This means that if the protein conformation changes, the microenvironment and UV-Vis absorption spectrum near the amino acid residues will also change. Therefore, a difference in the UV-Vis absorption spectrum of the protein after the addition of small molecules can be related to a change in the protein conformation.³⁴ The results show that amoxicillin affects the microenvironment of Trp, because this large hydrophobic amino acid is usually partially or fully buried within proteins or bound to ligands through hydrophobic interactions.⁴⁴ In addition, the Trp interaction can contribute significantly to the maximum wavelength of fluorescence by stabilizing or destabilizing the excited state and the ground state of the molecule. Therefore, the formation of bonds together with the excited state of Trp (stabilization) results in the release of energy in the form of heat, not light.⁷⁰,⁷¹

Trp together with other hydrophobic side chains of amino acids facilitates the formation of bonds with a significantly reduced level of ground state polarity compared to Trp itself. Consequently, the maximum wavelength of Trp fluorescence varies depending on the different bonds formed during the excited and ground states. The surface accessibility of the Trp residues and their interactions with other amino acid residues contribute to this variability.³⁴,³⁷ Thus, stronger bonds together with the excited state resulted in a shift toward longer wavelengths (redshift), whereas stronger bonds with the ground state led to a shortening of the emission waveform (blue shift).⁷¹

Analysis of molecular docking

To get a better understanding of the interactions of amoxicillin with Bf-LF, molecular docking was performed through AutoDock Tools v. 1.5 by calculating the best conformation of the ligand molecule and protein based on the lowest value of free energy binding. Figure 7 shows the best energy ranking result of the binding mode between amoxicillin and Bf-LF.

Figure 7
Molecular modeling study predicted orientations of the binding conformation of amoxicillin with the 3D-structure of Bf-LF (PDB ID: 1CE2).

The contribution of polar interactions of the hydroxyl group of the amoxicillin aromatic ring binding to arginine-Arg-342 and the binding of the amino group of amoxicillin to asparagine-Ans-330, with calculated bond lengths of 2.5 and 2.4 Å, respectively, may be observed in the simulation of the Bf-LF-amoxicillin (Figure 7). The results of the simulation also suggest that amino acid residues (Arg 342, Ans 330, glutamine-Gln 339, valine Val 338, alanine Ala 335, threonine-Thr 334, Glu 333, leucine Leu 331, isoleucine-Ile 127, Ile 231, Arg 130, Ala 337) are direct or indirect involved in the interaction of Bf-LF with amoxicillin.

Multiple studies¹⁴,²⁴,³⁴ report the antibacterial efficacy of bovine LF against mastitis pathogens. Bovine LF has shown various antibacterial effects on bacteria strains isolated from bovine milk.¹⁴ Intramammary infusion of bovine LF has also been tested for its impact against bovine mastitis, showing mild antibacterial activity. Formulations combining bovine LF with antibiotics increased the antibacterial effect of the antibiotics which contributes to reducing the amount of antibiotics needed to achieve a similar effect.²⁴,⁷² After optimizing the minimum inhibitory concentration, formulations of bovine LF with antibiotics showed a synergism effect against drug resistant pathogens.¹⁴ Aiming at a better understanding of the coupling between protein and antibiotics, spectroscopic studies of the interaction of bovine LF with tetracycline hydrochloride led to modifications in the LF conformation and function.³⁴ The study presented here shows the interaction of Bf-LF with amoxicillin resulting in modification on the protein conformation. These promising findings support the need for more extensive studies on the effect of formulations of Bf-LF-amoxicillin for the treatment of buffalo mastitis.

Conclusions

The fluorescence quenching and UV-Vis spectra indicated the formation of Bf-LF-amoxicillin complex and the conformation changes on the protein molecule. Fluorescence quenching spectra and UV-Vis spectra of the Bf-LF preparation indicated interaction of Bf LF amoxicillin generating a new complex, involving a static quenching mechanism with the main binding force being electrostatic. The results were further verified by in silico molecular docking experiments. Amoxicillin not only changed the structure of Bf-LF but also changed its original hydrophobicity, leading to a decrease in the hydrophobicity of Bf-LF. The results of the Bf LF amoxicillin binding are highly promising because it opens the possibility for further studies on the antibacterial activity of Bf-LF-amoxicillin complex.

Acknowledgments

We greatly appreciate the financial support by CAPES, the Brazilian Federal Agency for Support and Evaluation of Graduate Education for the scholarship granted to EFS. We also thank Mr Alberto de Gusmão Couto of the Castanha Grande farm in São Luiz de Quintunde for providing buffalo milk for the experiments.

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Edited by

• Editor handled this article:
  Ivo M. Raimundo Jr. (Associate)

Publication Dates

• Publication in this collection
  17 Jan 2025
• Date of issue
  2025

History

• Received
  08 May 2024
• Accepted
  10 Dec 2024