Protein-Resistant Property of Egg White Ovomucin under Different pHs

Oct 2, 2018 - E-mail: [email protected]., *Tel: +1-(780)492 1044. Fax: +1-(780)492 2881. E-mail: [email protected]. Cite this:J. Agric. Food Chem...
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Protein-resistant Property of Egg White Ovomucin under Different pHs and Ionic Strengths Xiaohong Sun, Jun Huang, Hongbo Zeng, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03905 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of Agricultural and Food Chemistry

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Protein-resistant Property of Egg White Ovomucin under Different pHs and

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Ionic Strengths

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Xiaohong Sun1,3, Jun Huang2, Hongbo Zeng2*, and Jianping Wu1*

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1

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Edmonton, AB, Canada,T6G 2P5

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2

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T6G 2V4

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3

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Department of Agricultural, Food & Nutritional Science, University of Alberta,

Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada

College of Food and Biological Engineering, Qiqihar University, Qiqihar,

Heilongjiang 161006, China

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*Corresponding authors:

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1

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4-10 Ag/For Centre, Department of Agricultural, Food & Nutritional Science,

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University of Alberta, Edmonton, AB, Canada, T6G 2P5 (telephone 1-780- 492-6885;

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fax 1-780- 492-4265; e-mail: [email protected])

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2

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Department of Chemical and Materials Engineering, University of Alberta, Edmonton,

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AB T6G 1H9, Canada. E-mail: [email protected];

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Fax: +1-(780)492 2881; Tel: +1-(780)492 1044

Dr. Jianping Wu

Dr. Hongbo Zeng

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Dr. Xiaohong Sun was responsible for literature search required for the study, experimental

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design, performing experiments, data analysis, and writing the manuscript. Dr. Jun Huang

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contributed to performing experiments, analyzing data, and manuscript revision of surface

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force measurements and atomic force microscope imaging parts. 1

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Abstract

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Ovomucin is a mucin-type glycoprotein accounting for 3.5% (w/w) of total egg

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white proteins. The purpose of the study was to explore the potential of ovomucin as a

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protein-resistant material. Using bovine serum albumin (BSA) as a model protein,

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ovomucin decreased the fluorescence intensities of the adsorbed BSA from

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10.90±2.18 to 0.67±0.75, indicating its protein-resistant property. To understand the

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underlying mechanism, pure repulsive forces between ovomucin and model proteins

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(e.g., BSA and ovomucin) at various pHs (2.0, 6.0 and 7.2) and ionic strengths (0.1,

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10, and 150 mM NaCl) were measured using a surface forces apparatus. Further

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studies by using atomic force microscope imaging, zeta potential and dynamic light

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scattering suggested that the protein-resistant property of ovomucin was mainly

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attributed to strong electrostatic and steric repulsions between protein layers. This

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work has demonstrated that ovomucin has anti-fouling potential with broad

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applications in the areas of food processing industry and biomedical implants.

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Key words: ovomucin, protein-resistant property, electrostatic and steric repulsions,

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pH, ionic strength

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Introduction

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Undesired accumulation of proteins on surfaces is a serious issue affecting

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numerous applications, such as food processing industry, biosensors, and biomedical

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implants

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processing results in decreased operating efficiency, less run times, increased

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possibility of biofilm formation and food contamination, and increased operating

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costs 3. Additionally, protein adsorption on surface reduces the sensitivity of biosensor

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and decreases the efficacy of biomedical implants. Furthermore, it may also result in

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undesirable host responses, such as blood coagulation, thrombus formation, platelet

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activation

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and subsequent biofilm formation since the protein layer could serve as a conditioning

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film 1. The device-related infections due to pathogenic bacteria adherence and biofilm

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formation on medical implants (such as catheters or prosthetic joints) are known to

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cause a high incidence of revision surgeries, and sometimes fatality

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from food safety, medical and economic point of view, development of protein-

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resistant surface to inhibit protein adsorption and subsequently prevent bacterial

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colonization is imperative.

1, 2

. For example, protein adsorption on stainless steel surfaces during food

1, 2, 4

. Protein adsorption also plays a vital role in the bacterial colonization

5, 6

. Therefore,

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The typical approach to prevent protein adsorption is to coat the substrate surface

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with a protein-resistant material that resists the non-specific interactions with proteins

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7

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based surfaces, zwitterionic polymer-based surfaces, and bactericidal polymer-based

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surfaces

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for inhibition of protein adsorption

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autoxidation in the presence of oxygen and forms aldehyde groups that may react with

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proteins, which makes PEG lose protein-resistant property 1, 14. Thus, it is necessary to

. Many protein-resistant surfaces have been explored, such as amphiphilic polymer-

8-11

. Poly(ethylene glycol) (PEG) based materials are the most widely used 8, 12, 13

. However, PEG is susceptible to

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identify alternative substances with protein-resistant property. The mucous layer covering epithelial cells functions as a natural anti-fouling 15

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surface to prevent undesirable adhesion to host tissues

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composed of water and mucins, a member of heavily glycosylated and gel-forming

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high molecular-weight glycoproteins 16. Therefore, much attention has been directed

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towards exploring the potential of mucin as an anti-fouling coating, especially bovine

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submaxillary mucin (BSM). BSM coatings could suppress cells and bacteria adhesion

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to biomaterials, which indicated its potential to serve as an anti-fouling surface in

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biomedical applications 17-19. Massive production of BSM is however impractical due

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to limited availability and high cost.

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. This layer is mainly

Egg is an easily available and relatively cheap commodity. Egg white contains 20, 21

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approximately 3.5% (w/w) of ovomucin, a mucin-type glycoprotein

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has similar structures as mammalian mucins: it has a long linear protein chain with a

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randomly coiled structure and the carbohydrate chains are attached to the protein core,

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which exhibits a ‘‘bottle brush’’ configuration 22; Ovomucin has a molecular weight

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of 23,000 kDa and subunits are linked by disulfide bonds 23. Ovomucin consists of a

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carbohydrate-poor (α-ovomucin) and a carbohydrate-rich (β-ovomucin) subunit,

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containing 11-15% and 50-57% (w/w) of carbohydrate, respectively 24, 25. On average,

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ovomucin contains 33% (w/w) of carbohydrate, including mannose (Man), galactose

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(Gal), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), N-

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acetylneuraminic acid (Neu5Ac) and sulfated saccharides 24, 26. Its structural similarity

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to mammalian mucins suggests that ovomucin may possess protein-resistant property.

. Ovomucin

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The objectives of this study were (1) to test the protein-resistant property of egg

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white ovomucin using bovine serum albumin (BSA) as a model protein to indicate the

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anti-fouling potential of ovomucin (2) to reveal the protein-resistant mechanism 4

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through direct surface force measurements between ovomucin and model proteins (i.e.,

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BSA and ovomucin) at different pHs and ionic strengths using a surfaces force

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apparatus (SFA)

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responsible for the protein-resistant property by characterizing surface morphology

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and determining zeta potential and average hydrodynamic size of proteins.

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Materials and Methods

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as well as (3) to further unraveling the molecular interactions

Materials and chemicals

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Fresh eggs from White Leghorn laid within 24h from the Poultry Research

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Centre of the University of Alberta (Edmonton, AB, CA) were used for extraction of

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ovomucin. Coomassie Brilliant Blue (CBB) R-250 was purchased from Bio-Rad (Bio-

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Rad Laboratories, Inc., Hercules, CA). 3-aminopropyltriethoxysilane (APTES) (98%)

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was obtained from Alfa Aesar (Ward Hill, MA, USA). Sodium chloride, bovine serum

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albumin (BSA), sodium chloride, sodium phosphate monobasic, sodium phosphate

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dibasic, sodium dodecyl sulfate (SDS), albumin-fluorescein isothiocyanate (FITC)

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conjugate and β-mecaptoethanol were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Hydrochloric acid was bought from Fisher Scientific Inc. (Fisher Scientific,

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Ottawa, ON, CA). Standard proteins (ovalbumin, ovomucoid, and lysozyme) were

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provided by Neova Technologies Inc. (Abbotsford, BC, CA). Mica sheets (ruby mica

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blocks, grade 1) were purchased from S&J Trading Inc. (Floral park, NY, USA).

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Milli-Q water was prepared by the Milli-Q water supply system (Millipore

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Corporation, Billerica, MA, USA), which was filtered through 0.2 µm PTFE filters

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(Mandel Scientific Company Inc., Guelph, ON, CA) prior to use.

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Extraction of ovomucin from egg white

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Ovomucin was extracted by the method established by our lab 29. Briefly, crude

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ovomucin was firstly precipitated by 100 mM of NaCl solution at pH 6.0. This slurry 5

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was kept in a cold room (4 ºC) overnight and then centrifuged at 15,300 g for 10 min

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at 4 ºC (Beckman Coulter, Inc., Fullerton, CA, USA). The precipitate was collected

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and re-suspended in 500 mM NaCl solution (pH 6.0). After stirring for 4 h, the

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suspension was settled at 4 ºC overnight. Finally, the precipitate was separated by

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centrifugation at 15,300 g for 10 min at 4 ºC (Beckman Coulter, Inc., Fullerton, CA,

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USA), dialyzed for 24 h, freeze-dried and stored at -20 ºC until use.

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Determination of the purity of ovomucin by gel filtration chromatography

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The purity of the extracted ovomucin was measured by a High-load 16/60 gel

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filtration column (Superdex 200 preparatory grade) coupled with Fast Performance

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Liquid Chromatography (FPLC) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden)

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as reported

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sodium phosphate buffer at pH 7.0 containing 50 g/L of SDS and 10 mL/L of β-

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mecaptoethanol. The injection volume was 3 mL, and ovomucin was eluted with 100

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mM phosphate buffer (pH 7.0) containing 5 g/L of SDS and 1 mL/L of β-

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mecaptoethanol at a flow rate of 1 mL/min, which was monitored by a UV detector at

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280 nm. Since there is no commercial ovomucin standard, the purity of ovomucin was

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calculated by subtracting the amount of other proteins (ovalbumin, ovomucoid, and

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lysozyme).

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. Ovomucins was dissolved at a concentration of 5 g/L in 100 mM of

Determination of the protein-resistant property of ovomucin in vitro

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The adsorption of BSA on an ovomucin-coated surface of the 96-well

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polystyrene microplate was tested to validate the protein-resistant property of

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ovomucin. Firstly, 200 µL of ovomucin (about 100 µg/mL) in 5 mM phosphate buffer

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with 150 mM NaCl at pH 7.2 was coated on the polystyrene surface for 24 h at room

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temperature. After washing with phosphate buffer, 200 µL of BSA-FITC (fluorescein

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isothiocyanate) conjugate at the concentration of 200 µg/mL was applied and 6

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incubated for 24 h at 37 ºC. The polystyrene surface was rinsed lightly with 200 µL of

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phosphate buffer twice, then the fluorescence intensity was applied to indicate the

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amounts of BSA adsorbed on the surface, which was determined by SpectraMax M3

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(Molecular devices, Sunnyvale, CA, USA) with an excitation at 495 nm and an

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emission at 520 nm. The adsorption of BSA on the non-coated polystyrene surface

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was measured as control 30.

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Ovomucin solution preparation

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Ovomucin was firstly suspended at the concentration of 5 mg/mL in 5 mM

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phosphate buffer with 150 mM NaCl at pH 7.2 and magnetically stirred overnight at

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1,200 rpm. Then the suspension was centrifuged at 5, 331 g for 15 min twice and the

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final ovomucin concentration in supernatant was diluted to 88 µg/mL, which was

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determined at 280 nm by a Nanodrop 1000 spectrophotometer (NanoDrop

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Technologies, Inc., Wilmington, DE, USA).

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

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SDS-PAGE was performed by using 4-20% ready-to-use gels (Bio-Rad

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Laboratories, Inc., Hercules, CA) to test the compostions of the above ovomucion

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solution, and the loaded amount of sample was 10 µg per well. Protein bands in the

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gel were stained with CBB R-250 for visualization.

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Atomic force microscopy (AFM) imaging

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The morphology of the ovomucin/BSA coated APTES-mica surface, and BSA

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deposited ovomucin-APTES-mica surface were characterized with an atomic force

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microscope (MFP-3D, Asylum Research, Santa Barbara, USA) under the acoustic

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tapping mode. Silicon cantilevers (Bruker Nano) with a nominal resonance frequency

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of 300−400 kHz and spring constant of ~40 N/m were used for imaging

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Preparation of ovomucin and BSA substrates was following the procedure described

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as above.

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Surface force measurement by surface forces apparatus (SFA) Surface preparation for surface force measurement

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Surface force measurements were mainly conducted based on three types of

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surfaces: 3-aminopropyltriethoxysilane (APTES)-coated mica, ovomucin coated on

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APTES-mica (ovomucin substrate), and BSA coated APTES-mica (BSA substrate).

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Mica surfaces were freshly cleaved in the laminar flow hood. APTES is an amino

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silane that is routinely employed for charge reversal or to create coupling layers on

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oxide surfaces

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amount of ovomucin and BSA proteins on mica. The APTES modified mica was

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prepared by a vapour deposition method which has been reported previously

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Briefly, fresh cleaved mica was treated by water plasma (40W, Harrick Plasma, USA)

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for creating more hydroxyl groups and then the treated substrate was placed in

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aplastic desiccator (diameter of 230 mm, Bel-Art Scienceware) with a small cap filled

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with 50 µL of APTES. Then the desiccator was kept under vacuum (100 mTorr) for

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12 hours to allow APTES vapor deposition. Later, those APTES treated mica were

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kept under vacuum for another 24 h for stabilization. To prepare ovomucin and BSA

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substrates, 500 µL of 88 µg/mL of ovomucin or 200 µg/mL of BSA solution was

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dipped onto the APTES treated mica, incubated for 20 min at 23 ºC, washed with

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Milli-Q water.

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32, 33

. Here, the APTES coating was applied to enhance the adsorption

33, 34

.

Surface force measurements

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SFA has been widely used to measure the interaction forces as a function of

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absolute separation distance between two surfaces with the force sensitivity down to

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10 nN and the distance resolution down to ~0.1 nm under different vapor/solvent 8

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conditions.

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interaction forces between ovomucin and different substrates (i.e., APTES coated-

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mica, BSA and ovomucin substrates) in aqueous solutions. In a typical SFA

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experiment, two back-silvered muscovite mica surfaces (thickness 1-5 µm, Grade #1,

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S&J Trading, USA) were glued onto cylindrical glass disks of radius R = 2 cm using

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an epoxy glue. After coating with desired protein layers, the two disks were mounted

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in the SFA chamber in a crossed-cylinder configuration, the interaction of which is

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equivalent to a sphere of the same radius interacting with a flat surface based on the

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Derjaguin approximation when the surface separation D is much smaller than R36.

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Then buffer solutions with different pHs (i.e., pH 2.0, 6.0, and 7.2) and ionic strengths

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(0.1 mM, 10 mM, and 150 mM) were injected between the two surfaces, respectively.

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The interaction force F between the curved surfaces at different pHs and ionic

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strengths was measured as a function of absolute surface separation distance D (it is

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worth noting that the zero distance is defined as APTES-APTES contact), which was

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determined in real-time based on an optical technique called multiple beam

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interferometry by using fringes of equal chromatic order (FECO). All the force

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measurements were conducted at 23 oC.

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In this work, the SFA technique was used to measure the

Particle size and zeta potential measurements

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The zeta potentials of ovomucin and BSA in solutions of different pHs and ionic

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strengths were determined at 23 ºC by a Zetasizer Nano ZSP (Malvern Instruments

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Ltd., Malvern, UK). The average hydrodynamic size was measured by dynamic light

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scattering (DLS) using the same Zetasizer Nano ZSP. The concentration of BSA was

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200 µg/mL, which was same as that in SFA measurements. Ovomucin was solubilized

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in the buffers with different pHs and ionic strengths according to the method

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introduced in the section of ovomucin solution preparation. 9

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Statistical analysis

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All experiments were conducted in triplicate and the results of particle size, zeta

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potential and fluorescence intensity of adsorbed BSA were expressed as mean ±

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standard deviation (SD). The surface force measurements were conducted on at least

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two different positions of the same pair of surfaces and the measurements were

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repeated using two pairs of surfaces prepared independently for the same

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experimental condition. All statistical analysis was carried out using IBM SPSS

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statistics 19 (SPSS Inc, USA), and significant differences were defined at a 5% level

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(P