Calcium Binding Capacity Research Articles

Preparation, identification and screening of anti-osteoporosis milk-derived peptides: Intervention effects in osteoporosis rats

To identify milk-derived peptides with both antioxidant and calcium absorption activities in combating osteoporosis, we employed a comprehensive screening approach that included virtual enzymatic hydrolysis, molecular docking, and cellular experiments using osteoblasts. Under the optimal conditions for dual-enzyme hydrolysis, the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging rate and soluble calcium binding capacity of the milk-derived peptides were 19.69% and 0.6965 μg/mL, respectively. Six peptide segments, namely KEDVPSER, HKEMPFPK, YPSYG, EDVPSE, VPQLE, and IPAVF, were identified through UPLC-Q-Exactive Orbitrap MS and molecular docking for further validation. Among the peptides, YPSYG significantly promoted the proliferation of MC3T3-E1 cells both with and without CaCl2 (P < 0.05), increasing proliferation by 38.27% and 20.67%, respectively, compared to the control group. Additionally, YPSYG significantly improved proliferation after H2O2-induced oxidative damage (P < 0.05), with a 38.23% higher rate than the model group. Compared with rats in the osteoporosis model group, YPSYG significantly enhanced serum alkaline phosphatase (ALP) and N-terminal propeptide of type I procollagen in rats (s-PINP) levels and decreased tartrate-resistant acid phosphatase (TRAP) levels (P < 0.05). Furthermore, milk-derived peptides and YPSYG significantly increased the bone weight index, maximum load, and bending energy of the femur and tibia in osteoporotic rats (P < 0.05). Additionally, these peptides significantly reduced the number of osteoclasts in the metaphysis of the femur and tibia in osteoporotic rats and alleviated microstructural damage. This study confirmed that milk-derived peptides, including YPSYG, effectively promoted bone formation and improved bone microstructure in osteoporotic rats. These findings provided a foundation for developing functional foods for elderly bone health.

Glycated walnut meal peptide‑calcium chelates (COS-MMGGED-Ca): Preparation, characterization, and calcium absorption-promoting

This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptide‑calcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptide‑calcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.

Effect of extraction method on the calcium binding capacity of faba bean globulin fractions at various pH

Faba bean ingredients are rich in proteins and good sources of calcium (Ca), although containing phytic acid (PA) molecules. PA, a polyphosphate compound, can affect the bioavailability of minerals/proteins through complex formation. This study evaluates the impact of two extraction processes, Alkaline Extraction-IsoElectric Precipitation (AE-IEP) and Sequential Extraction (SE), on the ability of faba bean globulin systems to bind added calcium ions. Increasing concentrations of CaCl2 were introduced into 2.5% (w/v) protein dispersions at pHs 4.5, 5.5, 6.5, and 7.5, and free Ca monitored. Near the isoelectric point of globulin (pH ∼ 4–5), Ca binding capacity was found to be low. At higher pHs, significant Ca chelation occurred, initially attributed to free PA binding sites, resulting in the formation of insoluble complexes and subsequent protein precipitation. The AE-IEP globulin fraction exhibited a higher Ca binding capacity than the SE globulin, attributed to its higher PA and lower initial Ca concentrations.

Enhancement of Calcium Chelating Activity in Peptides from Sea Cucumber Ovum through Phosphorylation Modification.

Recently, phosphorylation has been applied to peptides to enhance their physiological activity, taking advantage of its modification benefits and the extensive study of functional peptides. In this study, water-soluble peptides (WSPs) of sea cucumber ovum were phosphorylated in order to improve the latter's calcium binding capacity and calcium absorption. Enzymatic hydrolysis methods were screened via ultraviolet-visible absorption spectroscopy (UV-Vis), the fluorescence spectrum, and calcium chelating ability. Phosphorylated water-soluble peptides (P-WSPs) were characterized via high-performance liquid chromatography, the circular dichroism spectrum, Fourier transform infrared spectroscopy (FTIR), UV-Vis spectroscopy, surface hydrophobicity, and fluorescence spectroscopy. The phosphorus content, calcium chelation rate and absorption rate were investigated. The results demonstrated that phosphorylation enhanced the calcium chelating capacity of WSPs, with the highest capacity reaching 0.96 mmol/L. Phosphate ions caused esterification events, and the carboxyl, amino, and phosphate groups of WSPs and P-WSPs interacted with calcium ions to form these bonds. Calcium-chelated phosphorylated water-soluble peptides (P-WSPs-Ca) demonstrated outstanding stability (calcium retention rates > 80%) in gastrointestinal processes. Our study indicates that these chelates have significant potential to develop into calcium supplements with superior efficacy, bioactivity, and stability.

Preparation of Calcium-Binding Peptides Derived from Mackerel (Scomber japonicus) Protein and Structural Characterization and Stability Analysis of Its Calcium Complexes.

The purpose of this study was to prepare mackerel peptides (MPs) with calcium-binding capacity through an enzyme method and to investigate the potential role they play in improving the bioavailability of calcium in vitro. The calcium-binding capacity, degree of hydrolysis (DH), molecular weight (MW), and charge distribution changes with the enzymolysis time of MPs were measured. The structural characterization of mackerel peptide-calcium (MP-calcium) complexes was performed using spectroscopy and morphology analysis. The results showed that the maximum calcium-binding capacity of the obtained MPs was 120.95 mg/g when alcalase was used for 3 h, with a DH of 15.45%. Moreover, with an increase in hydrolysis time, the MW of the MPs decreased, and the negative charge increased. The carboxyl and amino groups in aspartic (Asp) and glutamate (Glu) of the MPs may act as calcium-binding sites, which are further assembled into compact nanoscale spherical complexes with calcium ions through intermolecular interactions. Furthermore, even under the influence of oxalic acid, MP-calcium complexes maintained a certain solubility. This study provides a basis for developing new calcium supplements and efficiently utilizing the mackerel protein resource.

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1–2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated “GE” motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8±1.8mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6±0.6mg calcium/g protein with a high association constant of 420M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Calcium absorption by Alaska pollock surimi protein hydrolysate promotes osteoblast differentiation.

The calcium-binding capacity and osteoblast proliferation and differentiation were studied in Alaska pollock surimi hydrolysate (APSH) using a system that mimics the gastrointestinal digestive system. Evaluation of the calcium absorption-promoting ability of APSH revealed that the best calcium-binding ability was achieved after hydrolysis with a combination of pepsin, α-chymotrypsin, and trypsin, and separation into <3kDa (APSH-I), 3-5kDa (APSH-II), 5-10kDa (APSH-III), and <10kDa (APSH-IV) fractions. Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis confirmed that the hydrolysate and calcium ions formed a complex. Comparison of the calcium absorption capacity using Caco-2 cells showed that calcium absorption was promoted by these hydrolysates. Measurement of the osteoblast activation revealed higher alkaline phosphatase activity, collagen synthesis, and mineralization effect for the low-molecular-weight hydrolysate (LMH) than for the other hydrolysates. In addition, LMH promoted the expression of osteocalcin, osteopontin, and bone morphogenetic protein-2 and -4, which are hormones related to bone formation. Expression of the Runx2 transcription factor, which regulates the expression of these hormones, also increased. These results suggest that Alaska pollock surimi protein hydrolysates prepared using a system that mimics gastrointestinal hydrolysis may result in better osteoblast proliferation and bone health than those prepared using other proteases.

Evaluating the capability of soybean peptides as calcium ion carriers: a study through sequence analysis and molecular dynamics simulations.

Calcium homeostasis imbalance in the body can lead to a variety of chronic diseases. Supplement efficiency is essential. Peptide calcium chelate, a fourth-generation calcium supplement, offers easy absorption and minimal side effects. Its effectiveness relies on peptide's calcium binding capacity. However, research on amino acid sequences in peptides with high calcium binding capacity (HCBC) is limited, affecting the efficient identification of such peptides. This study used soybean peptides (SP), separated and purified by gel chromatography, to obtain HCBC peptide (137.45 μg mg-1) and normal peptide (≤95.78 μg mg-1). Mass spectrometry identified the sequences of these peptides, and an analysis of the positional distribution of characteristic amino acids followed. Two HCBC peptides with sequences GGDLVS (271.55 μg mg-1) and YEGVIL (272.54 μg mg-1) were discovered. Molecular dynamics showed that when either aspartic acid is located near the N-terminal's middle, or glutamic acid is near the end, or in cases of continuous Asp or Glu, the binding speed, probability, and strength between the peptide and calcium ions are superior compared to those at other locations. The study's goal was to clarify how the positions of characteristic amino acids in peptides affect calcium binding, aiding in developing peptide calcium chelates as a novel calcium supplement.

Novel Calcium-Binding Peptide from Bovine Bone Collagen Hydrolysates and Its Potential Pro-Osteogenic Activity via Calcium-Sensing Receptor (CaSR).

This paper aims to explore the osteogenic activity and potential mechanism of the peptide-calcium chelate, and provides a theoretical basis for peptide-calcium chelates as functional foods to prevent or improve osteoporosis. In this research, a novel peptide (Phe-Gly-Leu, FGL) with a high calcium-binding capacity is screened from bovine bone collagen hydrolysates (CPs), calcium binding sites of which mainly included carbonyl, amino and carboxyl groups. The FGL-Ca significantly enhances the osteogenic activity of MC3T3-E1 cells (survival rate, differentiation, and mineralization). The results of calcium fluorescence labeling and molecular docking show that FGL-Ca may activate calcium-sensing receptor (CaSR), leading to an increase in intracellular calcium concentration, then enhancing osteogenic activity of MC3T3-E1 cells. Further research found that FGL-Ca significantly promotes the mRNA and protein expression levels of CaSR, transforming growth factor β (TGF-β1), TGF-β-type II receptor (TβRII), Smad2, Smad3, osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegrin (OPG), and collagen type I (COLI). Subsequently, in the signal pathway intervention experiment, the expression levels of genes and proteins related to the TGF-β1/Smad2/3 signaling pathway that are promoted by FGL-Ca are found to decrease. These results suggest that FGL-Ca may activate CaSR, increase intracellular calcium concentration, and activate TGF-β1/Smad2/3 signaling pathway, which may be one of the potential mechanisms for enhancing osteogenic activity.

The effect of nitric oxide donation on the severity of mitochondrial disfunction to the renal tissue in cardiopulmonary bypass simulation: an experimental study

INTRODUCTION: Acute kidney injury is one of the common complications in cardiosurgical operations with cardiopulmonary bypass (CPB). A number of studies have shown that donation of exogenous nitric oxide (NO) reduces episodes of аcute kidney injury. However, subcellular mechanisms of realization the nephroprotective properties of NO remain unknown. OBJECTIVE: To study the safety of the technology of plasma-chemical synthesis of nitric oxide and to evaluate the effect of the delivery resulting nitric oxide on mitochondrial damage to the renal tissue in the simulation of cardiopulmonary bypass. MATERIALS AND METHODS: Experiment included 12 rams of the Altai breed. Animals were divided into 2 groups: 6 animals were modeled CPB; 6 animals were simulated CPB with NO delivery. Mitochondrial damage was assessed by calcium-binding capacity and transmembrane potential of mitochondria 1 h after weaning from the CPB. The safety of the NO delivery according to the proposed method was assessed by the concentration of nitrogen dioxide on inspiration, the level of methemoglobin. The efficiency of NO delivery according to the proposed method was assessed by the level of stable NO metabolites: endogenous nitrite, nitrate and total concentration of NO metabolites. RESULTS: In the group of animals with NO delivery the average level of transmembrane potential of mitochondria was (171.66 ± 20.41 vs 126.66 ± 18.61; p = 0.00256) and calcium-binding capacity of mitochondria was (1466.66 ± 216.02 vs 866.66 ± 216.02; p = 0.000712) of renal parenchyma. Methemoglobin levels above the recommended thresholds in clinical practice were not recorded in the CPB+NO group. The values of total concentration of NO metabolites and nitrate in the CPB+NO group compared to the CPB group are statistically significantly higher, p = 0.00006; p = 0.0035, respectively. CONCLUSIONS: Plasma-chemical synthesis of nitric oxide is a safe technology, and the use of the resulting nitric oxide in cardiopulmonary bypass leads to a decrease in the severity of mitochondrial dysfunction in the kidney parenchyma.

Heat-induced changes of milk protein concentrate suspensions as affected by addition of calcium sequestering salts and shearing

Calcium sequestering salts (CSS) such as disodium hydrogen phosphate (DSHP) and trisodium citrate (TSC) can be used to improve the heat stability and solubility of milk proteins during thermal processing. Shearing is also an inherent part of food processing that can alter protein properties and functionality. The heat stability of milk proteins was investigated under combined temperatures (90 °C/5 min; 121 °C/2.6 min) and shear (100, 1000, 1500 s−1) with 10, 20, or 30 mm DSHP or TSC. The calcium-binding capacity of individual CSS predominantly determined the impact of shear. With DSHP, the effect of shear was more pronounced at high temperatures and shear rates than with TSC. The significant influence of shear on casein micelles in DSHP-containing dispersions suggested that DSHP's lower calcium binding affinity minimised micellar disruption and led to a pronounced shear impact. Shear combined with heat considerably impacts the milk system in the presence of CSS.

Phosphorylation modification of bovine bone collagen peptide enhanced its effect on mineralization of MC3T3-E1 cells via improving calcium-binding capacity

This study aimed to investigate the effect of phosphorylation modification of collagen peptide on its calcium-binding capacity and pro-mineralization activity. In this study, collagen peptide (Leu-Thr-Phe, LTF) and phosphorylated LTF (P-LTF) were synthesized and further chelated with calcium ions. The results showed that phosphorylation of LTF significantly enhanced its calcium-binding capacity. Spectra analysis revealed that the calcium-binding sites of P-LTF were mainly carbonyl, carboxyl, and phosphate groups. Molecular docking further demonstrated that the phosphate group introduced by phosphorylation enhanced the calcium-binding capacity of LTF by ionic bonds and coordination bonds. The stability analysis results suggested that intestinal fluid could repair the peptide-calcium complex destroyed by gastric fluid. The cell experiment displayed that P-LTF-Ca significantly improved the mineralization activity of MC3T3-E1 cells, and the order of effective influence was P-LTF-Ca > LTF-Ca > P-LTF > LTF. This study provided the theoretical basis for the potential application of phosphorylation modification in improving bone health.

Purification, Identification, Chelation Mechanism, and Calcium Absorption Activity of a Novel Calcium-Binding Peptide from Peanut (Arachis hypogaea) Protein Hydrolysate.

A novel calcium-binding peptide was purified from peanut protein hydrolysate using gel filtration chromatography and identified using HPLC-MS/MS. Its amino acid sequence was determined as Phe-Pro-Pro-Asp-Val-Ala (FPPDVA, named as FA6) with the calcium-binding capacity of 15.67 ± 0.39 mg/g. Then, the calcium chelating characteristics of FPPDVA were investigated using ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, particle size, and zeta potential. The results showed that FPPDVA interacted with calcium ions, the chelation of calcium ions induced FPPDVA to fold and form a denser structure, the calcium-binding sites may mainly involve oxygen atoms from the carboxyl residues of Asp and Ala, and Phe possessed contact energy and carbonyl residues of Val. Microstructure analysis showed that FPPDVA-calcium chelate exhibited a regularly ordered and tightly aggregated sheets or block structures. Additionally, FPPDVA-calcium chelate had good gastrointestinal digestive stability and thermal stability. The results of everted rat intestinal sac and Caco-2 cell monolayer experiments showed that FPPDVA-calcium chelate could promote calcium absorption and transport through the Cav1.3 and TRPV6 calcium channels. These data suggest that FPPDVA-calcium chelate possesses the potential to be developed and applied as calcium supplement.

Preparation, structure characterization, and stability analysis of peptide-calcium complex derived from porcine nasal cartilage type II collagen.

Porcine nasal cartilage type II collagen-derived peptides (PNCPs) may be complexed with calcium to provide a highly bioavailable, low-cost, and effective calcium food supplement. However, the calcium-binding characteristics of PNCPs have not yet been investigated. In the present study, calcium-binding peptides were derived from porcine nasal cartilage type II collagen and the resulting PNCPs-Ca complex was characterized. The study reveals that the calcium-binding capacity of PNCPs is closely related to enzymatic hydrolysis conditions. The highest calcium-binding capacity of PNCPs was observed at a hydrolysis time of 4 h, temperature of 40 °C, enzyme dosage of 1%, and solid-to-liquid ratio of 1:10. Scanning electron microscopy and energy dispersive X-ray spectroscopy revealed that the PNCPs had a pronounced capacity for calcium binding, with the PNCPs-Ca complex exhibiting a clustered structure consisting of aggregated spherical particles. Fourier-transform infrared spectroscopy, fluorescence spectroscopy, X-ray diffraction, dynamic light scattering, amino acid composition, and molecular weight distribution analyses all indicated that the PNCPs and calcium complexed via the carboxyl oxygen and amino nitrogen atoms, leading to the formation of a β-sheet structure during the chelation process. In addition, the stability of the PNCPs-Ca complex was maintained over a range of pH values consistent with those found in the human gastrointestinal tract, facilitating calcium absorption. These research findings suggest the feasibility of converting by-products from livestock processing into calcium-binding peptides, providing a scientific basis for the development of novel calcium supplements and the potential reduction of resource waste. © 2023 Society of Chemical Industry.

Calcium-binding properties, stability, and osteogenic ability of phosphorylated soy peptide-calcium chelate.

Bioactive peptides based on foodstuffs are of particular interest as carriers for calcium delivery due to their safety and high activity. The phosphorylated peptide has been shown to enhance calcium absorption and bone formation. A novel complex of peptide phosphorylation modification derived from soybean protein was introduced, and the mechanism, stability, and osteogenic differentiation bioactivity of the peptide with or without calcium were studied. The calcium-binding capacity of phosphorylated soy peptide (SPP) reached 50.24 ± 0.20 mg/g. The result of computer stimulation and vibration spectrum showed that SPP could chelate with calcium by the phosphoric acid group, carboxyl oxygen of C-terminal Glu, Asp, and Arg, and phosphoric acid group of Ser on the SPP at a stoichiometric ratio of 1:1, resulting in the formation of the complex of ligand and peptide. Thermal stability showed that chelation enhanced peptide stability compared with SPP alone. Additionally, in vitro results showed that SPP-Ca could facilitate osteogenic proliferation and differentiation ability. SPP may function as a promising alternative to current therapeutic agents for bone loss.

Electrocardiographic substrates of arrhythmias in patients with end-stage and chronic kidney diseases: a case–control study

BackgroundCardiovascular disease (CVD) is the most common cause of death in patients with renal diseases. Cardiac arrhythmia and sudden cardiac death are particularly important, and the burden is higher in patients on hemodialysis. The aim of this study is to compare specific ECG changes as markers of arrhythmias in patients with CKD and patients with end-stage renal disease (ESRD); all without clinically manifest heart disease, with normal control subjects.ResultsSeventy-five ESRD patients on regular hemodialysis, 75 patients with stage 3–5 CKD and 40 healthy control subjects were included. All candidates were subjected to thorough clinical evaluation and laboratory tests including serum creatinine, glomerular filtration rate calculation, serum potassium, magnesium, calcium, phosphorus, iron, parathyroid hormone, and total iron binding capacity (TIBC). Resting twelve-lead ECG was done to calculate P wave dispersion (P-WD), corrected QT interval, QTc dispersion, Tpeak-Tend interval (Tp-e), and Tp-e/QT. Patients with ESRD had a significantly higher QTc dispersion (p < 0.001) and P-WD (p = 0.001) when compared to the other 2 groups. In the ESRD group, males had a significantly higher P-WD (p = 0.045), insignificantly higher QTc dispersion (p = 0.445), and insignificantly lower Tp-e/QT ratio (p = 0.252) as compared to females. Multivariate linear regression analysis for ESRD patients showed that serum creatinine (β = 0.279, p = 0.012) and transferrin saturation (β = − 0.333, p = 0.003) were independent predictors of increased QTc dispersion while ejection fraction (β = 0.320, p = 0.002), hypertension (β = − 0.319, p = 0.002), hemoglobin level (β = − 0.345, p = 0.001), male gender (β = − 0.274, p = 0.009) and TIBC (β = − 0.220, p = 0.030) were independent predictors of increased P wave dispersion. In the CKD group, TIBC (β = − 0.285, p = 0.013) was an independent predictor of QTc dispersion while serum calcium (β = 0.320, p = 0.002) and male gender (β = − 0.274, p = 0.009) were independent predictors of Tp-e/QT ratio.ConclusionsPatients with stage 3–5 CKD and those with ESRD on regular hemodialysis exhibit significant ECG changes that are considered substrates for ventricular as well as supraventricular arrhythmias. Those changes were more evident in patients on hemodialysis.

Phosphorylation modification of tilapia skin gelatin hydrolysate and identification and characterization of calcium-binding peptides

In present study, complex protease was utilized to prepare tilapia skin gelatin hydrolysate (TSGH), and the phosphorylation conditions of TSGH were optimized using calcium-binding capacity. The phosphorylation degree of TSGH under the optimal conditions was 0.55%. The calcium-binding capacities of TSGH and phosphorylated TSGH were 44.21 and 62.87 µg/mg, respectively. Phosphorylation remarkably improved the calcium-binding capacity of TSGH. Forty-four peptides and 14 phosphopeptides were identified in phosphorylated TSGH, and SGPAGPK and its phosphopeptide, (p)SGPAGPK, were screened. The calcium chelates of SGPAGPK and (p)SGPAGPK were examined using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and mass spectrometry (MS). Phosphorylation of SGPAGPK effectively increased its calcium-binding capacity. SEM results indicated that SGPAGPK-Ca and (p)SGPAGPK-Ca were formed as new compounds. FTIR and MS results indicated that the carboxyl and phosphate groups of Ser and the amino group of Lys may be the calcium chelation sites.

Crystal Structure Analysis of Sarcoplasmic-Calcium-Binding Protein: An Allergen in Scylla paramamosain.

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp70 and Asp18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.

Calcium-chelating improved zein peptide stability, cellular uptake, and bioactivity by influencing the structural characterization

To improve the calcium intake, stability, and functional properties, calcium-chelating zein peptide (Ca-ZP) was developed from zein. The preparation conditions, structural characterization, stability, cellular uptake, antioxidant and antihypertensive activities of the Ca-ZP were investigated compared to the zein peptide (ZP). The highest calcium content and yield of Ca-ZP were 85.71 % and 42.91 %, respectively. After binding ZP carboxyl and amino groups with Ca2+, Ca-ZP was formed, confirmedby structural analyses. Moreover, Ca-ZP exhibited good stability at wide pHs, temperatures, and under-simulated gastrointestinal digestion in vitro, as well as antioxidant and ACE inhibitory capacity with the IC50 values of DPPH scavenging, hydroxyl radical scavenging, and ACE inhibitory activities of 0.48, 0.96, and 0.49 mg/mL respectively. Ca-ZP gastrointestinal digestive fluid showed higher calcium transport and absorption capacity than CaCl2 digestivefluid. Overall, Ca-ZP possessed high calcium-binding capacity, calcium absorption bioavailability, stability, and bioactivity. This work provides a promising approach for preparing calcium-chelating zein peptides, which are applicable as calcium supplements, antioxidants, and antihypertensive products.