Protein-Resistant Property of Egg White Ovomucin under Different pHs

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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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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

ACS Paragon Plus Environment


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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

3



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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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.


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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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27, 28, 35

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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

209

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

Protein-Resistant Property of Egg White Ovomucin under Different pHs

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