Novel peptide with a specific calcium-binding capacity from whey protein hydrolysate and the possible chelating mode

Article abstract of DOI:10.1021/jf502412f

A novel peptide with a specific calcium-binding capacity was isolated from whey protein hydrolysates. The isolation procedures included diethylaminoethyl (DEAE) anion-exchange chromatography, Sephadex G-25 gel filtration, and reversed-phase high-performance liquid chromatography (HPLC). A peptide with a molecular mass of 237.99 Da was identified by liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS), and its amino acid sequence was confirmed to be Gly-Tyr. The calcium-binding capacity of Gly-Tyr reached 75.38 μg/mg, increasing by 122% when compared to the hydrolysate complex. The chelating interaction mode between the Gly-Tyr and calcium ion was investigated, indicating that the major binding sites included the oxygen atom of the carbonyl group and nitrogen of the amino or imino group. The folding and structural modification of the peptide arose along with the addition of the calcium ion. The profile of ¹H nuclear magnetic resonance (NMR) spectroscopy demonstrated that the electron cloud density around the hydrogen nucleus in the peptide changed was caused by the calcium ion. The results of ζ potential showed that the Gly-Tyr-Ca chelate was a neutral molecule in which the calcium ion was surrounded by the specific binding sites of the peptide. Moreover, thermogravimetry-differential scanning calorimetry (TG-DSC) and calcium-releasing assay revealed that the Gly-Tyr-Ca chelate exerted excellent thermal stability and solubility in both acidic and basic conditions, which were beneficial to calcium absorption in the gastrointestinal tract of the human body and, therefore, improved its bioavailability. These findings further the progress in the research of whey protein, suggesting the potential in making peptide-calcium chelate as a dietary supplement.

Full text of DOI:10.1021/jf502412f

Article
pubs.acs.org/JAFC
Novel Peptide with a Specific Calcium-Binding Capacity from Whey
Protein Hydrolysate and the Possible Chelating Mode
Lina Zhao, Qimin Huang, Shunli Huang, Jiaping Lin, Shaoyun Wang,* Yifan Huang, Jing Hong,
and Pingfan Rao
College of Bioscience and Biotechnology, Fuzhou University, Fuzhou, Fujian 350002, People’s Republic of China
ABSTRACT: A novel peptide with a specific calcium-binding capacity was isolated from whey protein hydrolysates. The
isolation procedures included diethylaminoethyl (DEAE) anion-exchange chromatography, Sephadex G-25 gel filtration, and
reversed-phase high-performance liquid chromatography (HPLC). A peptide with a molecular mass of 237.99 Da was identified
by liquid chromatography−electrospray ionization/mass spectrometry (LC−ESI/MS), and its amino acid sequence was
confirmed to be Gly-Tyr. The calcium-binding capacity of Gly-Tyr reached 75.38 μg/mg, increasing by 122% when compared to
the hydrolysate complex. The chelating interaction mode between the Gly-Tyr and calcium ion was investigated, indicating that
the major binding sites included the oxygen atom of the carbonyl group and nitrogen of the amino or imino group. The folding
1
and structural modification of the peptide arose along with the addition of the calcium ion. The profile of H nuclear magnetic
resonance (NMR) spectroscopy demonstrated that the electron cloud density around the hydrogen nucleus in the peptide
changed was caused by the calcium ion. The results of ζ potential showed that the Gly-Tyr−Ca chelate was a neutral molecule in
which the calcium ion was surrounded by the specific binding sites of the peptide. Moreover, thermogravimetry−differential
scanning calorimetry (TG−DSC) and calcium-releasing assay revealed that the Gly-Tyr−Ca chelate exerted excellent thermal
stability and solubility in both acidic and basic conditions, which were beneficial to calcium absorption in the gastrointestinal tract
of the human body and, therefore, improved its bioavailability. These findings further the progress in the research of whey
protein, suggesting the potential in making peptide−calcium chelate as a dietary supplement.
KEYWORDS: whey protein, calcium-binding peptide, purification, characterization, chelating mode
absorbed in the human body. The transportation of metal
elements in chelate depends upon ligands. The revolving
mechanism of small peptides is much different from that of
amino acids. In comparison to amino acids, the absorption of
small peptides has many merits, such as consuming little
energy, accelerating transport speed, and carriers not being easy
to saturate.13 Hydrolyzed whey peptides, obtained from
proteolytic digestion, have shown considerable capacity in
incorporating with divalent ions, such as calcium, iron, etc.14,15
The chelating complex chelated between whey peptides and
calcium ion can promote calcium absorption in the human
body and, therefore, improve its bioavailability. Peptide-
chelated calcium can probably be a suitable candidate as a
supplement to improve calcium absorption in the gastro-
intestinal tract of the human body.
The purpose of this study was to investigate the purification
of specific calcium-binding peptide derived from whey protein
hydrolysate and the possible chelating mode. To evaluate the
chelation mode between the purified calcium-binding peptide
and calcium ion, structural characterization methods were
applied in detail. The study would be of significance in using
the hydrolyzed peptides from whey protein as calcium-binding
peptide ingredients in functional foods. The physiological
INTRODUCTION
■
Whey protein is a mixture of globular proteins isolated from
whey as the byproduct in the cheese production process, and it
releases a variety of bioactivities through the appropriate
protease hydrolysis.1,2 As one of the good protein sources,
whey protein and its hydrolysates can be used as a food
ingredient, nutritional supplement, or functional intensifying
agent. It is well-reported that whey protein has calcium-binding
sites that can bind with calcium ion, such as β-LG, α-LA, and
lactoferrin.3,4 Moreover, α-LA has especially strong calcium-
binding sites.5,6 However, studies on the purification of specific
calcium-binding peptide derived from whey protein hydrolysate
and the possible chelating mode are scarcely reported.
Calcium is an essential nutrient in the body, and adequate
calcium intake is related with the low risk of osteoporosis,
hypertension, colon caner, obesity, and kidney stones.7 The
intake of calcium could increase the bone density in children,
and it is essential among the middle-aged and aged to prevent
osteoporosis.8,9 With the increase in the population of the aged
throughout the world, there is a growing interest in developing
calcium supplementary medicine to prevent and treat bone
disease.10 The ionized calcium has served as the main calcium
supplements for humans in recent years.11 However, the
disadvantage of ionized calcium is that it is prone to form
calcium phosphate deposition in a basic intestine environ-
ment.12 As a result, the bioavailability of dietary calcium is
severely lowered. The organic calcium supplement has been
becoming one of popular research topics. Both amino acid
chelate and small peptide calcium complex can be directly
Received: May 24, 2014
Revised: September 29, 2014
Accepted: September 29, 2014
© XXXX American Chemical Society
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Sodium phosphate was used as the calcium-binding buffer, which
could react with calcium ion and peptide, separately, and show
obviously different reactions. Calcium ion could combine with
phosphate to form calcium phosphate precipitation, while peptide
could take calcium ion from precipitation. As a result, peptide−Ca
chelate was a water-soluble substance, and a soluble calcium content in
the supernatant represented the calcium content associated with the
peptide.16 The calcium contents in the supernatant were determined
using a colorimetric method with ortho-cresolphthalein complexone
reagent.17 The absorbance at 570 nm was determined after adding the
working solution to the sample. The experiments were performed in
triplicate, and values were expressed as the mean standard deviation
(SD).
Characterization of the Purified Calcium-Binding Peptide.
Formation of the Peptide−Calcium Chelate. Peptide−calcium
chelate was the chelating component of the reaction between
dipeptide Gly-Tyr and calcium ion. The calcium-binding chelate was
prepared by adding 5 mL of 1% (w/v) CaCl2 into 20 mL of 2.5% (w/
v) calcium-binding peptide solution. The reaction was placed in a
controlled water bath with constant agitation (100 rpm) at 37 °C for 2
h after the pH of the solution was adjusted to 7.0 by the addition of 0.1
M NaOH. Then, the mixture was added with absolute ethanol (9
times of solution volume) to remove free calcium and centrifuged at
10000g for 10 min, and the precipitates was lyophilized for analysis.
Ultraviolet (UV) Absorption Spectroscopy Assay. The UV spectra
of the calcium-binding peptide and peptide−calcium chelate were
recorded over the wavelength range from 190 to 400 nm by a UV−vis
spectrophotometer (UV-2600, UNICO Instrument Co., Ltd.,
Shanghai, China) as the method described by Chen et al.18 The
calcium-binding peptide of 0.2 mg/mL was prepared. The peptide−
calcium chelate was prepared by adding 0, 2, 4, 6, 8, and 10 μM CaCl2
to 0.2 mg/mL calcium-binding peptide solution, separately. The mixed
solution reacted at room temperature for 30 min.
Fluorescence Spectra Analysis. Fluorescence spectra were
measured to monitor conformational changes in the peptide induced
by calcium chelation using the F-4600 fluorescence spectrophotometer
(Hitachi Co., Japan). The excitation wavelength was 280 nm, and
emission wavelengths between 290 and 360 nm were recorded.
Fourier Transform Infrared Spectroscopy (FTIR) Measurement. A
total of 1 mg of calcium-binding peptide powder or peptide−calcium
chelate was ground evenly with 100 mg of dry KBr under infrared light
to the size below 2.5 μm. Then, the transparent KBr piece was made at
60 MPa. To examine whether the variation in pellet thickness causes
significant interference in the measured spectra, three different pellets
were prepared from the same sample and their FTIR spectra were
compared. The average of these three spectra was then used for
analysis. The FTIR spectra were recorded using an infrared
spectrophotometer (360 Intelligent, Thermo Nicolet Co., Waltham,
MA) over a wavenumber region between 4000 and 400 cm−1 at a
resolution of 4 cm−1, and the sample spectra were background-
subtracted using a spectrum collected in the absence of a peptide−
calcium chelate sample. Therefore, the physical variation in the FTIR
spectra caused by the KBr technique had been removed prior to the
FTIR spectra analysis. A total of 64 scans were recorded per sample in
the FTIR spectra, and the peak signals in the spectra were analyzed
using OMNIC 8.2 software (Thermo Nicolet Co., Madison, WI).
1H Nuclear Magnetic Resonance (NMR) Spectroscopy Assay. The
peptide−calcium chelate and calcium-binding peptide (0.5 mg) were
dissolved in 500 μL of deionized water, separately. A total of 50 μL of
deuterium oxide (D2O) was added after the pH of the solution was
adjusted to 6.5. The samples were transferred into 5 mm NMR tubes
and subjected to NMR analysis with a Bruker Avance III spectrometer
(Bruker Biospin, Rheinstetten, Germany).
activity of calcium-binding peptides may be used as one kind of
food additive that prevents bone disorders.
MATERIALS AND METHODS
■
Materials. Whey protein (WPC80, containing 80.5% protein) was
kindly provided by Hilmar Corporation (Batch 20111107, Hilmar,
CA). The commercial proteases, Flavourzyme (EC. 3.4.11.1, 2 × 106
units/g) and Protamex (EC. 3.4.24.28, 1.5 × 106 units/g), were
purchased from Novo (Novozymes, Denmark). Toyopearl DEAE-
650M and Sephadex G-25 were offered by Amersham Pharmacia
(Amersham Pharmacia Co., Uppsala, Sweden). All of the other
chemicals and solvents were of analytical grade.
Hydrolysis of Whey Protein. A total of 5% (w/v) whey protein
solution was denatured in 80 °C for 20 min, and the pH was adjusted
to 7.0. The sample was hydrolyzed using Flavourzyme and Protamex
(2:1, w/w) with a substrate/enzyme ratio of 25:1 (w/w) at 49 °C for 7
h. Hydrolysate was heated at boiling water for 10 min to inactive the
enzyme and cooled to room temperature. The mixture was
subsequently centrifuged at 16000g for 20 min, and then the
supernatant named whey protein hydrolysate (WPH) was lyophilized
and stored at −20 °C for subsequent investigation.
Purification and Identification of Calcium-Binding Peptide.
Ion-Exchange Chromatography on Diethylaminoethyl (DEAE). The
peptide that could bind with calcium and form peptide−calcium
chelate was defined as the calcium-binding peptide. The slurry of
Toyopearl DEAE-650M was packed in a column (20 × 2.5 cm) and
then equilibrated at 5 column volume (CV) of 20 mM Tris-HCl buffer
(pH 9.0) as the equilibrating buffer. Afterward, 100 mg of the
lyophilized hydrolysates that had been through 0.45 μm filter film was
dissolved in 10 mL of the same buffer (pH 9.0) and loaded on the
column. Then, after washing with the equilibrating buffer, the collected
peak was labeled as the non-absorbed fraction. The bond peptides
were eluted by a gradient elution with the same buffer containing 0−
0.5 M NaCl. The flow rate was 0.5 mL/min; fraction volume was 5
mL/tube; elution was monitored at 214 nm; all peaks were collected;
and calcium-binding capacities of the fractions were determined.
Gel Filtration on Sephadex G-25. The fraction with the highest
calcium-binding capacity was pooled and lyophilized; 200 mg of the
sample was dissolved in 5 mL of deionized water and loaded onto a
Sephadex G-25 column (100 × 2.0 cm), which had previously been
equilibrated with deionized water, and then eluted with deionized
water at a flow rate of 0.3 mL/min. The eluate was monitored by
measuring the absorbance at 214 nm. After calcium-binding capacity
was determined, the fraction with the highest activity was pooled and
lyophilized.
High-Performance Liquid Chromatography (HPLC) on C18. The
lyophilized sample collected from G-25 was dissolved in distilled water
approximately equivalent to 30 mg/mL and purified by semi-
preparative reversed-phase high-performance liquid chromatography
(RP-HPLC) on C18 reversed silica gel chromatography (Gemini 5 μm
C18, 250 × 10 mm, Phenomenex, Inc., Torrance, CA). Elution was
performed on solution A [0.05% trifluoroacetic acid (TFA) in water]
and solution B (0.05% TFA in acetonitrile) with a gradient of 0−30%
B at 1.0 mL/min for 50 min. The elution was monitored at 214 nm,
and the absorption peaks were fractionated for measuring the calcium-
binding activity. The injection volume was generally 200 μL.
Identification of the Purified Calcium-Binding Peptide. The
molecular mass and amino acid sequence of the purified calcium-
binding peptide were determined using liquid chromatography−
electrospray ionization/mass spectrometry (LC−ESI/MS, Delta Prep
4000, Waters Co., Milford, MA) over the m/z range of 300−3000.
Calcium-Binding Capacity Assay. The calcium-binding capacity
was defined as the content of calcium (μg) bound with peptide (mg)
after the chelating reaction. Lyophilized whey protein hydrolysate was
dissolved in deionized water to be 1.0 mg/mL and mixed with 5 mM
CaCl2 in 0.2 M sodium phosphate buffer (pH 8.0). The solution was
stirred at 37 °C for 2 h, and pH was maintained at 8.0 with a pH
meter. The reaction mixture was centrifuged at 10000g at room
temperature for 10 min to remove insoluble calcium phosphate salts.
Differential Scanning Calorimetry (DSC) Analysis. Thermogravim-
etry (TG)−DSC simultaneous thermal analyzer (STA449C,
NETZSCH, Germany) was employed to measure the thermal
property. Hermetic pans with lyophilized powder samples (5 mg)
were heated from 50 to 600 °C at a programmed heating rate of 10
°C/min in a argon atmosphere. An empty pan was used as a reference.
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ζ Potential Measurement. A Malvern Nano ZS Zetasizer based on
dynamic light scattering was employed to measure the ζ potential. The
system works according to the phase analysis light scattering (PALS)
principle. The volume of all samples (3 mg) was 15 mL with
autotitration by 0.25 M NaOH or HCl. The samples were equilibrated
for 30 s before titration.
named F21, F22, F23, and F24 (Figure 2). F23 exerted the
most excellent calcium-binding capacity (Figure 2). The result
Calcium-Releasing Percentage Experiment. The calcium-releasing
percentage determination of peptide−calcium chelate and CaCl2 was
examined according to the method described by Wang et al.19
Peptide−calcium chelate and CaCl2 were dissolved in deionized water,
separately, equivalent to 10 μg/mL. The pH of these solutions was
adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. After incubation in a
shaking water bath for 2 h at 37 °C, the solutions were centrifuged in a
refrigerated centrifuge at 10000g for 10 min. The calcium amount in
the supernatant and the total calcium in the whole solution were
determined by the colorimetric method with an ortho-cresolphthalein
complexone reagent. The calcium releasing percentage was calculated
as follows:
calcium‐releasing percentage (%)
= Ca amount in supernatant (g)/total Ca in solution (g) × 100
Figure 2. Fractionation of the fraction F2 with the highest calcium-
binding activity from DEAE chromatography on a Sephadex G-25 gel
filtration chromatography column and the calcium-binding capacity of
the fractions F21−F24 separated from a Sephadex G-25 column.
Statistical Analyses. All data were presented as means (SDs)
conducted with three replicates. The statistical analysis was performed
using SPSS 16.0 software (SPSS, Inc., Chicago, IL). Analysis of
variance (ANOVA) was performed to determine the significance of
the main effects. A value of p < 0.05 was considered statistically
significant.
was consistent with some previous reports, which showed that a
lower molecular mass peptide exhibited a tendency to combine
with calcium ion.21,23 It is reported that the lowest molecular
weight peptide fraction (<1 kDa) had the higher calcium-
binding activity through ultrafiltration and small molecular
peptide fractions purified on a G-25 column possessed higher
calcium-binding capacity.21,22
The active peak F23 was pooled, dissolved in distilled water,
and loaded onto semi-preparative C18 RP-HPLC. The fraction
F23 was separated into 13 major distinct fractions (Figure 3A).
Each fraction was pooled, and calcium-binding capacity was
determined subsequently (Figure 3B). Among 13 distinct
fractions, fraction 7 showed the highest calcium-binding ability,
and then it was further fractionated by RP-HPLC on a C18
column. The elution profile showed that fraction 7 was
composed of five peaks, and fraction 2 (Figure 4A), one of the
eluted peaks, was the purifed peptide. The calcium-binding
capacity of fraction 2 reached 75.38 μg/mg (Figure 4B). The
chromatographic profile of fraction 2 on analytic C18 RP-
HPLC (Figure 5) showed only a single peak called WPH-A,
indicating the high purity of the isolated peptide.
RESULTS AND DISCUSSION
■
Purification and Identification of the Calcium-Binding
Peptide. Bioactive peptides are usually purified using a
combination of chromatographic techniques.20 Throughout
the purification process, calcium-binding activity of WPH was
assessed using the colorimetric method with an ortho-
cresolphthalein complexone reagent.17 Three fractions (F1,
F2, and F3) of the WPHs were divided on a DEAE anion-
exchange chromatography column (Figure 1). The calcium-
The amino acid sequence of WPH-A eluted from RP-HPLC
chromatography was identified by LC−ESI/MS (Figure 6).
According to the mass/charge ratio (m/z) of 238.99 Da of the
molecular ion peak ([M + H]+), the molecular mass of WPH-A
was confirmed to be 237.99 Da. The m/z of fragment ion y1
was 181.99 Da, which resulted from the relative molecular mass
of tyrosine in addition with one hydrogen atom. On the basis of
calculation in ESI ion molecular mass, one residue in WPH-A
was Tyr and the other one was Gly. Therefore, the animo acid
sequence was determined to be Gly-Tyr. The result was
confirmed by comparison to data from the National Center for
Biotechnology Information (NCBI) database.
Not only could the differences in the net charge and length
of peptides influence the extent of chelate formation with metal
ion, but also the different amino acid residues of peptides
affected their activities,14,24 including the amino acid
composition and sequence of the peptides.25 Chaud et al.14
reported that tyrosine seemed to bind with divalent metal
Figure 1. Fractionation on a DEAE anion-exchange chromatography
column and the calcium-binding capacity of the three fractions
separated from the DEAE column. The peptide was washed with the
binding buffer (0.02 M Tris−HCl buffer at pH 9.0) and eluted with a
linear gradient of 0−0.5 M NaCl in the same buffer.
binding capacity was noticeably higher in F2 and F3 compared
to F1 (Figure 1). Moreover, in comparison to WPH (33.92 μg/
mg), the calcium-binding capacity of F2 was significantly
improved (57.02 μg/mg).
Fraction F2 with the highest calcium-binding capacity was
further separated using size-dependent Sephadex G-25 gel
filtration chromatography resulting in four major fractions,
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Figure 5. Analytic RP-HPLC of the peak WPH-A for purify
identification. The column was washed with a linear gradient of 0−
10% acetonitrile containing 0.05% TFA.
Figure 3. (A) Fraction F23 from a Sephadex G-25 column and then
applied to a semi-preparative C18 RP-HPLC column. The column was
then washed with a linear gradient of 0−30% acetonitrile containing
0.05% TFA. (B) Calcium-binding capacity for the fractions 1−13 of
F23 separated from semi-preparative HPLC.
Figure 6. Identification of the amino acid sequence of the calcium-
binding peptide using LC−ESI/MS.
Alaska pollack containing Gly, Ser, and Val. Hoki bone
hydrolysates had a high affinity to calcium, and the major
animo acid composition of the peptide included Gly, Asp, and
Glu.28 An iron-chelating peptide with multi-glycine residues
was also purified from anchovy muscle protein.29 In this study,
a calcium-binding peptide was identified to contain Gly and Tyr
residues; both of them contributed to combine with divalent
minerals.
Characterization of the Purified Calcium-Binding
Peptide. UV Absorption Spectroscopy Assay. The UV spectra
of calcium-binding peptide Gly-Tyr showed an obvious
difference from that of the Gly-Tyr−Ca chelate (Figure 7). A
strong absorption band was observed at about 200 nm, which
could be regarded as the characteristics of the amide bond in
peptide. Another absorption peak at around 280 nm appeared
in both Gly-Tyr and Gly-Tyr−Ca chelate, resulting from the
characteristic UV absorption of Tyr. With the addition of
calcium ion to dipeptide Gly-Tyr, the intensity of the
absorption band of the amide bond became higher than that
of dipeptide alone. In the magnified UV spectra image, the red
shifts of the band, such as the red curve in Figure 7, could be
clearly observed and a small peak arose in the dipeptide−
calcium chelate.
Through observing the shifts and intensity changes of UV
bands, the UV absorption situation of the amide bond in
peptide and the side group in Tyr could be analyzed. The UV
spectra phenomenon was consistent with some other studies,
which demonstrated that the spatial structure with the chirality
of the chromospheres (CO and −COOH) and auxochromes
(−OH and −NH2) of peptides changed after incorporating
with the calcium ion.30,31 The main reason for the intensity
changes of the band indicated that the oxygen atom of the
Figure 4. (A) RP-HPLC of fraction 7 derived from preparative HPLC.
The column was then washed with a linear gradient of 0−10%
acetonitrile containing 0.05% TFA. (B) Calcium-binding capacity of
fractions 1−5 from RP-HPLC.
cations through oxygen of the phenolic hydroxyl group. The
phosphorylation of tyrosine and serine residues could provided
suitable binding sites for positively charged metals, such as
calcium, iron, or zinc.26 Moreover, the animo acid glycine was
considered as an organic ligand having the metal ion binding
function. Jung et al.27 purified a calcium-binding peptide from
D
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This result is consistent with that of the iron-chelating peptides
from anchovy (Engraulis japonicus) muscle protein.29 Reddi et
al.33 demonstrated that the ZnII binding energy was used to fold
the peptide in the zinc-binding reaction of a zinc-finger peptide.
As the concentration of CaCl2 increased, fluorescence
quenching became weaker than before. It meant that there
were not any spared binding sites in Gly-Tyr for the calcium
ion. This experimental evidence suggested that the structure of
functional binding sites in Gly-Tyr changed, resulting in the
formation of the Gly-Tyr−Ca chelate when CaCl2 reacted with
Gly-Tyr. The addition of metal ion induced the intrinsic
fluorescence quenching of protein or peptide, especially for the
oligo-peptide without thermodynamic structure stability.34
Generally, the calcium ion could cause fluorescence quenching
of the calcium-binding peptide, which likely contributed to the
decrease in the fluorescence intensity.19
FTIR Measurement. Changes in the characteristic FTIR
absorption peaks of some functional binding sites in peptide
could reflect the interaction of metal ion with organic ligand
groups in the peptide. To analyze calcium-ion-induced changes
in the structure of the calcium-binding peptide Gly-Tyr, the
FTIR technique was employed.
Figure 7. UV spectra of Gly-Tyr and Gly-Tyr−Ca chelate over the
wavelength range from 190 to 400 nm.
carbonyl group and nitrogen of the amino group in the peptide
bond with calcium.32 The appearance of a small peak reflected
the difference of relevant valence electron transition when more
calcium ion reacted with Gly-Tyr. All of these band changes
suggest that Gly-Tyr could bind with calcium ion and form the
Gly-Tyr−Ca chelate.
Figure 9 depicted the infrared spectrum of Gly-Tyr in the
absence and presence of calcium. There were significant
Fluorescence Spectra Analysis. The purified calcium-
binding dipeptide Gly-Tyr contained aromatic amino acid
Tyr, which could generate endogenous fluorescent at a proper
excitation wavelength. The fluorescence emission spectra of
Gly-Tyr was examined to monitor its structural modification
with CaCl2 treatment. Through fluorescence quenching, the
calcium binding sites could be speculated. The fluorescence
spectra of Gly-Tyr and Gly-Tyr−Ca chelate are shown in
Figure 8. When excited at 280 nm, Tyr in Gly-Tyr exhibited an
intrinsic fluorescence emission maximum at 310 nm. With the
increased calcium ion concentration, the endogenous fluo-
rescence decreased dramatically, which meant that calcium ion
had a significant effect on the Gly-Tyr structure. Especially,
apparent folding and conformation modification of Gly-Tyr
happened at the beginning of the addition with 5 μM CaCl2.
Figure 9. FTIR spectra of Gly-Tyr and Gly-Tyr−Ca chelate in the
regions from 4000 to 400 cm−1.
differences between Gly-Tyr and Gly-Tyr−Ca chelate. The
results implied that the wavenumbers (1674 and 1437 cm−1) of
the amide I band were shifted to lower frequencies (1585 and
1413 cm−1) after the addition of calcium, which stood for
infrared absorption of the carbanyl group caused by the
antisymmetric stretching vibration υas (COO−) and the
symmetric stretching vibration υs (COO−) of carboxylic acid
ions. Upon coordination with the Gly-Tyr−Ca chelate, the C
bands corresponding to the carboxyl groups at 1204 and 1139
cm−1 exhibited two weaker peaks in the fingerprint region. It is
reported that ferric chloride could bind with casein enzymatic
hydrolysates to form the Fe3+−peptide complex, and the chief
iron-binding site relates to the amide and carboxylate groups.14
When metal ions combined with ligand atoms, such as O, N,
and S, of organic compounds to form the chelate, the
absorption peaks because of vibration of the coordinate
bonds were typically located in the far-infrared region. The
Figure 8. Fluorescence emission spectra of the peptide Gly-Tyr at
various concentrations of Ca2+.
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Figure 10. 1H NMR spectral region from −0.30 to 10.00 ppm of Gly-Tyr and Gly-Tyr−Ca chelate (the red line means Gly-Tyr, and the green line
means Gly-Tyr−Ca).
1
Ca−O vibration band was located between 500 and 800 cm−1,
while the interaction between the peptide and calcium ion
resulted in a broadening and decreased intensity of the peaks;
even some band absorptions disappeared, which was in
agreement with the report by Chen et al.18 Absorption of a
high frequency at 3084 cm−1 referred to the antisymmetric
stretching vibration υas (N−H) and the symmetric stretching
vibration υs (N−H) of the N−H bond in the Gly-Tyr
spectrum. Absorption of a high frequency at 3200−3600
cm−1 referred to the possible Ph−O-H strech and H bonds of
hydration in the Gly-Tyr spectrum. With the coordination of
the calcium−peptide complex, the waveform and the
absorbance values had greatly changed. After the addition of
calcium, the chelation reaction resulted in a shift to a higher
wavenumber (3407 cm−1), which indicated that the electron
cloud density of N−H in Gly-Tyr became stronger because of
the inductive effect or dipole field effect.
The chemical shift information on H NMR could explain
the distribution of the electron cloud density around the
hydrogen nucleus among different substances; consequently,
the reaction situation between the purified calcium-binding
dipeptide Gly-Tyr and calcium ion could be revealed. A
declined electron cloud density along with a decreased
shielding effect and intensive resonant frequency led to the
movement of resonance signal peaks to a lower field region.
TG−DSC Analysis. The thermostability of samples was
investigated through DSC and TG thermal analysis. There were
remarkable differences of peak temperatures between dipeptide
Gly-Tyr and Gly-Tyr−Ca chelate (Figure 11). The TG−DSC
curve of Figure 11A showed that the thermal decomposition
reaction of Gly-Tyr was not single in the whole process of
weight loss. The thermal transitions at 179.40, 269.15, 359.99,
and 530.51 °C were accompanied by weight loss of 25.74,
14.23, 15.15, and 14.32%, respectively. These endothermic
peaks were caused by the C−N bond in Gly-Tyr. Whereas,
obviously high shift endothermic peaks were observed at
428.77, 462.05, 504.07, and 550.93 °C after calcium ion
treatment (Figure 11B). Meanwhile, TG of Gly-Tyr−Ca
showed a flat and decline curve with a mass loss of 80%,
which demonstrated that calcium-ion-processed Gly-Tyr
resulted in a great increase in the DSC peak temperature
accompanied by less and reduced weight loss, indicating that
the Gly-Tyr−Ca chelate exhibited distinct thermal stability.
This result suggested that Ca-processed dipeptide Gly-Tyr was
less sensitive to heat denaturation with a higher peak
temperature because of the formation of the Gly-Tyr−Ca
chelate compound and the thermal stability of Gly-Tyr−Ca
chelate was higher than that of Gly-Tyr alone.
These phenomena indicated that the interaction between
Gly-Tyr and calcium occurred via the carboxyl oxygen atoms
and amino group nitrogen atoms in the peptide, which were
similar to the calcium-binding peptides derived from soybean
protein hydrolysates.35
1H NMR Spectroscopy Assay. The low-field region, from
6.75 to 8.00 ppm, of the Gly-Tyr and Gly-Tyr−Ca NMR
spectrum (Figure 10) contained signals from aromatic group
tyrosine (6.75 and 7.10 ppm) and N−H of the amide bond
(8.00 ppm). The symmetric doublet in the region of 3.55−3.75
ppm corresponded to H spin coupling cracking in α-H of Gly
and Tyr. The signals at 3.00 and 2.75 ppm arose from two β-H
atoms of tyrosine. With the addition of the calcium ion, the
symmetric doublet signals of α-H in Gly-Tyr shifted to lower
parts per million (ppm) values, whose chemical shift was
approximately 0.025 ppm. The change in hydrogen atom spin
coupling cracking resonance signal peaks was caused by the
combination of Gly-Tyr and calcium, thereby the electron
density around the hydrogen nucleus in Gly-Tyr was affected.
ζ Potential. The principal of ζ potential was polarization of
the water that occurred in the interface of two phases and then
inducing that potential difference that existed in the phases.
Figure 12 presented the ζ potential values of the Gly-Tyr−Ca
chelate compared to Gly-Tyr without the calcium ion. The
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indicated that the addition of calcium improved the pH value of
Gly-Tyr, inducing a weaker charge, and made it prone to be an
neutral molecule (Figure 12B). Besides, the ζ potential ranged
from positive charge (19.3 mV) to negative charge (−13.8 mV)
when the calcium ion was added to dipeptide Gly-Tyr. Figure
12A also showed that the pI of Gly-Tyr was 4.46, while that of
Gly-Tyr−Ca was 3.35. The pI decreased with the calcium ion
treated on Gly-Tyr. In addition, the pH 3.34 of Gly-Tyr in
aqueous solution was closer to its pI 4.46 than that of Gly-Tyr−
Ca, which suggested that Gly-Tyr−Ca was more stable than
Gly-Tyr alone. In the same pH value range, the changes of the ζ
potential absolute value of Gly-Tyr−Ca were less than those of
Gly-Tyr and Gly-Tyr−Ca possessed a lower ζ potential
absolute value. The result indicated that Gly-Tyr−Ca existed
as a molecular structure in aqueous solution without two
different phases or double electrode layers. Through this
experimental evidence, it could be speculated that the calcium
ion was surrounded by the functional binding sites, such as the
carboxyl and amino groups, of Gly-Tyr by a coordinate bond
exhibiting an neutral molecule without being a form of
inorganic calcium, to enhance its stability in the gastrointestinal
tract.
Calcium-Releasing Percentage Assay. The calcium-releas-
ing percentage of the Gly-Tyr−Ca chelate and CaCl2 at
different pH values is shown in Figure 13. The solubility
Figure 11. Typical DSC thermograms of (A) Gly-Tyr and (B) Gly-
Tyr−Ca chelate.
Figure 13. Calcium-releasing percentage of the Gly-Tyr−Ca chelate
and CaCl2 at pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, separately.
between Gly-Tyr−Ca and CaCl2 was apparently different. For
both of them, the calcium-releasing percentage reduced with
the increase of pH. While at any pH value, the calcium-releasing
percentage of Gly-Tyr−Ca was obviously higher than that of
CaCl2. The calcium-releasing amount in the Gly-Tyr−Ca
chelate was relatively stable in the pH that ranged from 2.0
to 8.0, maintaining above 95%, whereas that of CaCl2 decreased
significantly from 93.94% at pH 2.0 to 78.48% at pH 8.0. The
result implied that calcium in Gly-Tyr−Ca had good solubility
whether at pH 2.0 or 8.0, which was similar to the pH values of
the human stomach or gastric environment; consequently,
precipitation could not form.36
Figure 12. ζ potential of (A) Gly-Tyr and (B) Gly-Tyr−Ca chelate.
initial pH of Gly-Tyr in aqueous solution was measured to be
3.34 (Figure 12A). It meant that dipeptide Gly-Tyr in aqueous
solution exhibited acid property. While in the Gly-Tyr−Ca
chelate, the initial pH 5.45 approached neutral pH 7.0, which
It is essential to discuss the dissolved status of the peptide−
calcium chelate in the human gastrointestinal tract because the
calcium ion probably reacted with phytic or oxalic acid to
become insoluble in the stomach and form Ca(OH)2 in the
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Figure 14. Hypothetical molecular structural formula of the Gly-Tyr−
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Characterization of phosphopeptide derived from bovine β-casein: An
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-591-22866375. Fax: +86-591-22866278. E-
mail: shywang@fzu.edu.cn.
■
(17) Gitelman, H. J. An improved automated procedure for the
determination of calcium in biological specimens. Anal. Biochem. 1967,
18, 521−531.
Funding
(18) Chen, D.; Liu, Z.; Huang, W.; Zhao, Y.; Dong, S.; Zeng, M.
Purification and characterisation of a zinc-binding peptide from oyster
protein hydrolysate. J. Funct. Foods 2013, 5, 689−697.
(19) Zhou, J.; Wang, X.; Ai, T.; Cheng, X.; Guo, H.; Teng, G.; Mao,
X. Preparation and characterization of β-lactoglobulin hydrolysate−
iron complexes. J. Dairy Sci. 2012, 95, 4230−4236.
This work was supported by the National Marine Public
Welfare Projects (201305022), the Fujian Natural Science
Foundation of China (2013J01132), the open funding of Fujian
Provincial Key Laboratory of Photocatalysis−State Key
Laboratory Breeding Base of China (038018), Natural Science
Foundation of China (31471623).
(20) Megías, C.; Pedroche, J.; Yust, M. M.; Giron
́
-Calle, J.; Alaiz, M.;
Millan, F.; Vioque, J. Affinity purification of copper-chelating peptides
́
Notes
from sunflower protein hydrolysates. J. Agric. Food Chem. 2007, 55,
6509−6514.
The authors declare no competing financial interest.
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of a hepta-peptide with iron binding activity from shrimp processing
by-products hydrolysates. Adv. J. Food Sci. Technol. 2012, 4, 207−212.
(22) Huang, G.; Ren, L.; Jiang, J. Purification of a histidine-
containing peptide with calcium binding activity from shrimp
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