An Exploration of the Calcium-Binding Mode of Egg White Peptide

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An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and in vitro Calcium Absorption Studies of Peptide-Calcium Complex Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, and Songyi Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03705 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Journal of Agricultural and Food Chemistry

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An Exploration of the Calcium-binding Mode of Egg White Peptide,

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Asp-His-Thr-Lys-Glu, and in vitro Calcium Absorption Studies of

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Peptide-Calcium Complex

4 Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, Songyi Lin*

5 6 7

National Engineering Research Center of Seafood, School of Food Science and

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Technology, Dalian Polytechnic University, Dalian 116034, P. R. China

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*

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Professor Songyi Lin

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No. 1 Qinggongyuan, Ganjingzi District

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National Engineering Research Center of Seafood, School of Food Science and

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Technology, Dalian Polytechnic University, Dalian, P.R. China, 116034

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Tel.: +86 18840821971

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Fax: +86 0411 86318655

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E-mail address: [email protected]

Corresponding author:

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1

ACS Paragon Plus Environment


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ABSTRACT:

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The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates

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and calcium ions was elucidated upon its structural and thermodynamics

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characteristics. The present study demonstrated that the DHTKE peptide could

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spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding

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site corresponded to the carboxyl oxygen, amino nitrogen and imidazole nitrogen

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atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex

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on improving the calcium absorption was investigated in vitro using Caco-2 cells.

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Results showed that the DHTKE-calcium complex could facilitate the calcium influx

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into the cytosol and further improve calcium absorption across Caco-2 cell

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monolayers by more than seven times when compared to calcium-free control. This

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study facilitates the understanding about the binding mechanism between peptides

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and calcium ions as well as suggests a potential application of egg white peptides as

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nutraceuticals to improve calcium absorption.

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KEYWORDS: egg white peptide, calcium binding, calcium absorption, Caco-2 cells

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INTRODUCTION

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Calcium is known as an essential mineral for human body to maintain bone health.1,2

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Bone loss, which results in metabolic bone diseases such as rickets and osteoporosis,3

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has been proved to be mainly related to insufficient calcium absorption. It has been

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reported that the amount of calcium absorbed by the body depends on the amount of

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soluble calcium in the duodenum and proximal jejunum.4 The solubility of calcium

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can be diminished due to the precipitation of calcium ions with antagonists such as

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oxalate, phytates and cellulose in the intestines.5,6

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Recently, there has been a great deal of interest in the enhancing effects of food

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substances (particularly peptides) on calcium solubility and subsequent calcium

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absorption. Binding calcium to peptides for preventing from calcium precipitation can

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effectively increase the absorption of calcium in the body.7,8 Currently,

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calcium-binding peptides have been found from various food sources, including hen

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egg yolk,9 cow milk casein,10 whey,12,14 soy,15 wheat germ,16 tilapia fish,17,18 Alaska

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pollock19,20 and shrimp processing byproducts.21 Casein phosphopeptides (CPPs) and

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phosvitin phosphopeptides can promote the absorption of calcium by chelating

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calcium with the phosphoserine residues. However, several peptides lacking

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phosphoserine residues also enhance intestinal calcium uptake, by binding calcium to

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the Asp and Glu residues.

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Asp, Glu, Ser, His, and Lys are the most frequently reported calcium-binding

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ligands. CPPs are originally found to have an ability to bind calcium ions and increase

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calcium absorption, which is exactly attributed to the role of the acidic sequence

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“Ser(P)-Ser(P)-Ser(P)-Glu-Glu”.22, 23, 24 Afterwards, Chen et al.18 reported that Asp,

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Gly and Glu were the most abundant amino acids in the calcium-binding peptide from

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tilapia scale protein. The calcium-binding peptides derived from soybean protein 3



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hydrolysates,15 bovine serum protein hydrolysates,25 and porcine blood plasma protein

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hydrolysates,26 also possessed Asp and Glu residues. Furthermore, peptides containing

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histidine residues have been reported to possess high affinity to calcium ions. Huang

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et al.21 isolated a histidine-containing tri-peptide, Thr-Cys-His, from shrimp

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processing byproducts to possess high calcium-binding capability, which might be

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responsible for the presence of His residue. As a matter of fact, the calcium-chelating

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activity of these amino acids could be ascribed to their specific groups, including

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carboxyl groups of Asp and Glu, the δ-N in the imidazole ring of His, and the ε-amino

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nitrogen of Lys. Most current researches have focused on the isolation and

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identification of calcium-binding peptides, and the exploration of specific amino acids

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and groups that contribute to calcium binding. More detailed studies are limited that

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characterize the stoichiometry, binding affinity and thermodynamics of calcium

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binding to purified peptides, or their biological properties through in vitro and in vivo

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studies.

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Our previous study isolated an egg white pentapeptide, Asp-His-Thr-Lys-Glu

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(DHTKE),33 which possesses the amino acids related to calcium binding. This study

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was designed to elucidate the binding mode between the DHTKE peptide and calcium

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ions upon its structural and thermodynamics characteristics, and investigate the effect

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of the DHTKE peptide on calcium absorption by human intestinal epithelial cells. The

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possible calcium-binding sites were investigated using analytical techniques such as

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ultraviolet absorption spectroscopy, Fourier transform infrared spectroscopy, and 1H

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nuclear magnetic resonance (NMR) spectroscopy. Isothermal titration calorimetry was

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applied to investigate the association constant and stoichiometry of the

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peptide-calcium complex. A Caco-2 cell model was applied to assess calcium

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absorption. This study could be conducive to better understand the binding 4


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mechanism between peptides and calcium ions, and suggests a potential application of

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egg white peptides as nutraceuticals to improve calcium absorption.

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MATERIALS AND METHODS

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Materials. An egg white pentapeptide, Asp-His-Thr-Lys-Glu (DHTKE), was

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synthesized in 98.85% purity by China Peptides Co., Ltd. (Shanghai, China). Eagle’s

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minimal essential medium, penicillin-streptomycin-neomycin (PSN) antibiotic

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mixture and trypsin-EDTA were obtained from Gibco (Burlington, Ontario). Fetal

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bovine serum (FBS) was supplied by PAN-Biotech (Bavaria, Germany). Fluo-3 AM

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was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin (from

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porcine pancreas) and pepsin (from porcine gastric mucosa) were obtained from Bio

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Basic Inc. (Toronto, Canada) and Sigma-Aldrich (St. Louis, MO), respectively.

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Preparation of the DHTKE-calcium Complex. For fabrication of the

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DHTKE-calcium complex, 3.6 mM of DHTKE peptide was mixed with 21.6 mM

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CaCl2 in 20 mL Milli-Q water. The binding reaction was performed at pH 8.0 and

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50 °C for 1 h under continuous stirring. Thereafter, absolute ethanol was added to the

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mixture to a final concentration of 90%, resting for 60 min to precipitate the

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complexes. After centrifugation at 12,000×g for 5 min, the precipitates was collected,

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freeze-dried and labeled as DHTKE-calcium.

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Ultraviolet-visible Absorption Spectroscopy. Ultraviolet-visible spectra were

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measured to testify the occurrence of the chelation reaction between peptide and

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calcium. 0.1 mg/mL of the DHTKE peptide solution was prepared and mixed with 0.2,

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0.4 or 0.8 mM CaCl2 to obtain the DHTKE-calcium complex. The ultraviolet-visible

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spectra of the DHTKE peptide and its calcium complex were measured within a

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wavelength range between 190 and 800 nm by using a UV-Vis spectrophotometer 5



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(Perkin Elmer, Salem, MA).

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Zeta Potential Determination. The zeta potential of the DHTKE peptide and

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DHTKE-calcium complex was measured using a Zetasizer Nano ZS90 particle size

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analyzer (Malvern Instruments Ltd., Malvern, UK), according to the method from the

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previous studies of Sun et al.11 1 mg/mL of the DHTKE peptide or DHTKE-calcium

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solution was added into an U-shaped cell. Subsequently, the cell temperature was

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maintained at 25 °C for 5 s, and all measurements was performed at 25 °C and

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repeated 12 times.

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Fourier Transform Infrared Spectroscopy. Freeze-dried DHTKE peptide or

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DHTKE-calcium powder (2 mg) was grinded evenly with 100 mg KBr under infrared

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light. The mixed powder was then compressed into a thin disc. The spectra between

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4000 and 400 cm-1 were performed through a Fourier transform infrared (FTIR)

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spectrometer (Perkin Elmer, Salem, MA) at a resolution of 4 cm-1. A total of 32 scans

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were recorded per sample in the FTIR spectra, and the analysis of FTIR spectra was

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performed using an OMNIC 8.2 software (Thermo Fisher Scientific Inc.)

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1

H Nuclear Magnetic Resonance (NMR) Spectroscopy. The 1H NMR spectra of

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the DHTKE peptide and DHTKE-calcium complex were determined by a Bruker

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AVANCE III 400 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany), using

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a modification of the methodology of Lin et al.27 with some modifications. 5 mg

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samples were dissolved in 600 µL of dimethyl-d6 sulfoxide solution containing 0.03%

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(v/v) tetramethylsilane (TMS), transferred into 5 mm NMR tubes and subjected to

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NMR analysis. A spectral width of 8012.8 Hz was used with a relaxation delay of 1.5

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s and a total of 16 scans were recorded.

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Isothermal Titration Calorimetry (ITC). ITC measurements were conducted at

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25 ± 0.2 °C by an Affinity ITC calorimeter (TA Instruments Ltd., New Castle, DE). 6


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The DHTKE peptide and CaCl2 were dispersed in Tris/HCl buffer (pH 8.0), passing

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through a 0.22-µm Millipore membrane. All samples were degassed before loading

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into the sample cell and injection syringe. Reactions were conducted by titrating 40

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consecutive CaCl2 solution (50 mM) into 5 mM of DHTKE peptide solution.

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Controlled trials were conducted by injecting CaCl2 solution into Tris/HCl buffer.

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Raw ITC data were fitted to an independent-site binding model using the Nano

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Analyze software by which stoichiometry (n), binding constant (K), and enthalpy (∆H)

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and entropy change (∆S) were calculated.

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Calcium Absorption Studies. Cell culture. Caco-2 cells were supplied by Cell

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Resource Center in Shanghai Institutes for Biological Sciences, the Chinese Academy

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of Sciences (Shanghai, China). The cells were cultured in Eagle’s minimal essential

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medium, supplemented with 20% FBS and PSN antibiotic mixture, and incubated at

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37 °C in a humidified 5% CO2 incubator. The differentiation of Caco-2 cells was

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achieved according to the well-established procedure29 consisting of successive

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sub-cultivations.

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Calcium imaging. Calcium influx into Caco-2 cells, which is expressed with

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calcium imaging, was measured as previously described by Perego et al.8 with some

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modifications. Caco-2 cells were seeded on 35-mm confocal dishes, and loaded with

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2.5 µM Fluo-3 AM, in Krebs-Ringer HEPES (KRH) solution (140.0 mM NaCl, 5.0

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mM KCl, 2.0 mM CaCl2, 0.55 mM MgCl2, 6.0 mM glucose and 10.0 mM HEPES,

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pH 7.4). After incubation at 37 ºC in darkness for 30 min, cells were washed in KRH

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solution and allowed to a further 30 min incubation for de-esterification of the

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fluorescent probe. Fluorescence analysis was performed by a Leica SP8 confocal

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laser-scanning microcopy (Leica Microsystems, Wetzlar, Germany) using a 40× (oil)

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magnification. The cells, maintained in 2 mL of KRH solution, were excited at a 7



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wavelength of 488 nm. The emission was recorded at 510 nm. Baseline calcium

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signal was monitored, and then 2 mmol/L of DHTKE-calcium complex or CaCl2 (100

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µL) was added to the 35-mm confocal dishes, followed by the measurement of

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cytoplasmic calcium signal. Finally, 100 µL of ATP (100 µmol/L) was added prior to

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the end of the assay.

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Establishment of Caco-2 cell monolayer model. Passage numbers between 30 and

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50 were applied in the establishment of Caco-2 cell monolayer model for calcium

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transport studies. Cells were seeded at a density of 1.5 × 105 cells/mL on 12-well

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transwell culture plates (Corning Inc., NY) with a polycarbonate membrane (12 mm

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diameter inserts, 0.4 µm pore size). The culture medium in the apical and basolateral

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sides was replaced every other day during the first week and then replaced daily until

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applied for calcium transport tests. Transepithelial electrical resistance (TEER) was

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measured every other day using a Millicell-ERS system (Millipore, Billerica, MA,

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USA) to assess the integrity of the cell monolayers.

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Calcium transport studies. The monolayers with TEER values above 400 Ω × cm2

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were applied for this experiment.30 The cell monolayers were gently rinsed twice with

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Hank’s balanced salt solution (HBSS, without calcium and magnesium) and then

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moved to a new plate containing 1.5 mL of HBSS buffer. Afterwards, different

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concentrations of DHTKE-calcium (0-6 mM) or 2 mM CaCl2 in 0.5 mL HBSS buffer

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(pH 7.4) were added to the apical side, and incubated at 37 °C for 2 h. 1.0 mL of

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sample was extracted from the basolateral side at 30, 60, 90, and 120 min, and then

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1.0 mL of HBSS buffer was added to the basolateral side in order to keep the volume

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constant.

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spectrophotometer (Hitachi Co., Tokyo, Japan). Calcium absorption was calculated

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according to Cao et al.31 by using the following equation:

Calcium

contents

were

determined

by

8


an

atomic

absorption

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n −1

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Bn = 1.5 × An + 1.0 × ∑ Ak

(1)

k =1

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where B n represents the transported calcium content in 1.5 mL of HBSS buffer in the

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basolateral side of each well at 30, 60, 90 and 120 min; 1.5mL is a constant,

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representing 1.5 mL of HBSS buffer in the basolateral side of each well; An represents

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the calcium concentration of the HBSS buffer in the basolateral side of each well at

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different time points; 1.0 is also a constant and represents the 1.0 mL of HBSS buffer

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collected from the basolateral side of each well in order to measure the calcium

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content; n is an independent variable. For the present study, it could be the number of

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1, 2, 3 or 4, representing time points 30, 60, 90, and120 min, respectively.

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Statistical Analysis. All experiments were conducted in triplicate, and data

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analysis was made by employing SPSS 18.0 software (SPSS Inc., Chicago, IL). A

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one-way ANOVA was used to evaluate the significant differences of data, with the

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confidence level set at P

An Exploration of the Calcium-Binding Mode of Egg White Peptide

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