Number Of Free Amino Groups Research Articles

Effects of κ-carrageenan addition and chlorogenic acid covalent crosslinking on protein conformation and gelling properties of soy protein hydrogels

This paper reports the effects of κ-carrageenan (KC) additions (0.6% w/v) and covalent crosslinking by chlorogenic acid (CA) on the protein conformation and gelling properties of soybean protein isolate (SPI) hydrogels. Treatment with KC and CA changed the conformation of the SPI and improved the antioxidant capacity and gel properties, as confirmed by fluorescence spectroscopy, Fourier-transform infrared spectroscopy, antioxidant capacity analysis, and rheology measurements. The addition of CA (0.01 g/g-SPI) decreased the number of free amino groups, free sulfhydryl content, tryptophan fluorescence intensity, and surface hydrophobicity of SPI by 44.22%, 10.05%, 30.47%, and 44.61%, respectively, and increased the DPPH and ABTS radical scavenging activities by 30.29% and 40.69%, respectively. The addition of KC (0.6% w/v) enhanced the DPPH radical scavenging activities of the SPI-CA (0.002 and 0.01 g/g-SPI) conjugates by 11.29–16.50%, and improved the viscoelasticity, water immobilization, density, and thermal stability of the SPI–CA gels.

Analysis of the Structure and Antigenicity in Ovalbumin Modified with Six Disaccharides Through Liquid Chromatography-High-Resolution Mass Spectrometry.

Melibiose, cellobiose, maltose, lactose, turanose, and isomaltulose were selected to be glycated with OVA. The number of free amino groups of OVA modified with different disaccharides decreased, and the secondary and tertiary structures of the modified OVA also changed greatly. Moreover, the glycation sites detected by HPLC-HCD-MS/MS were all on the sensitized epitopes of OVA, which reduced the binding ability of IgG and IgE of glycated OVA. In addition, the glycation sites with the highest DSP in different samples were located in the irregular coil region of OVA. Among the six disaccharides, the glycation reaction between melibiose and OVA was the most obvious. Through the analysis of disaccharide configuration, it was found that the glycation efficiency of the reducing disaccharide linked by a 1 → 6 glycoside bond was higher than that by a 1 → 4 glycoside bond, and reducing sugar with β type was better than that with α type. These findings would provide a theoretical reference for the use of different sugars in food production.

Determination of effective assay parameters on the activity of magnetite cross-linked invertase aggregates by personal glucose meter

The invertase enzyme has been immobilized onto various support materials to enhance operational stability, shelf life, and reusability compared to free ones. Among the immobilization methods, carrier-free immobilization, such as cross-linked enzyme aggregates (CLEAs), attracts attention. However, recovery of the CLEAs from the reaction environment by centrifugation or filtration is a great challenge. The use of magnetic nanoparticles (Fe3O4) can overcome these separation limitations of the aggregates. For this aim, in this study, the surface of magnetite nanoparticles was functionalized with a significant number of free amino groups through silanization reaction. After precipitation of these aggregates and then cross-linking to the functionalized structure, the resulting magnetite cross-linked invertase aggregates (MCLIAs) were obtained, having different enzyme and glutaraldehyde concentrations. Magnetic nanoparticles were characterized by SEM, VSM, and FTIR analysis. The assay parameters, both constructional and experimental ones on the activity of MCLIAs, were practically determined by a personal glucose meter (PGM). The results indicate that magnetite nanoparticles with superparamagnetic behaviour were successfully functionalized with amino groups. The activity results demonstrated that experimental parameters were more effective than constructional parameters. MCLIAs also exhibited maximum activity at pH 5. Furthermore, 30-min incubation time and 35 °C were the best activity assay conditions.

Частота выявления протеинурии у кошек с хронической болезнью почек

Proteinuria is the excretion of any type of protein in the urine in values exceeding the physiological norm (up to 0,010 g/l). Proteinuria is a marker and factor of kidney disease, leading to a decrease in life expectancy and quality of life in this group of patients. For the detection of abnormal urinary protein excretion, the gold standard of diagnosis is the determination of the urinary protein/creatinine ratio by a quantitative test. Semi-quantitative studies on test strips are of screening value, they are more sensitive to albumin due to the large number of free amino groups than globulins, this research method shows results at a protein concentration of more than 0,30 g/l. The etiology of proteinuria is multifactorial and the causes can be prerenal, renal and postrenal, and it is also worth considering the possibility of detecting functional proteinuria - increased physical activity, hyperthermia (fever), convulsions. Of particular clinical importance is true renal proteinuria, which may occur as a result of damage to the tubular apparatus, glomeruli and / or interstitium. Several studies have found that cats with chronic kidney disease and confirmed proteinuria have a shorter survival rate than cats with chronic kidney disease without proteinuria. Thus, detection and control of proteinuria is an important aspect in the diagnosis and management of cats with chronic kidney disease.

Effect of (E,E)‐2,4‐decadienal on Side‐Chain Modification, Conformation Change, and Aggregation of Bovine Serum Albumin

AbstractProtein modification by active aldehydes is associated with ageing and some chronic diseases, and it is closely related to food quality and safety. In this study, the modification of bovine serum albumin (BSA) using trans,trans‐2,4‐decadienal at various concentrations (0–10 mm) is investigated. The results show that the changes in the number of free amino groups and arginine residues, carbonyl formation, aggregation characteristics, and fluorescent lipofuscin‐like pigments (LFLP) fluorescence are more severe at (E,E)‐2,4‐decadienal concentrations of 4–10 mm, whereas the conformational changes are more significant at low concentrations (0.1–2 mm). In the BSA modified by 1–10 mm (E,E)‐2,4‐decadienal, the microenvironment of tryptophan residues to become more hydrophobic, the structure became compact, hydrophobic binding sites decreases, molecular weight and particle size increases, and covalently cross‐linked heterogeneous aggregates increases. At the same time, the relationship between the oxidation parameters and samples is explained using principal component analysis and hierarchical cluster analysis.Practical Applications: (E,E)‐2,4‐decadienal is highly electrophilically active with biomacromolecules. However, compared to the widely studied malonaldehyde (MDA), acrolein (ACR), 4‐hydroxynonenal (HNE), and 4‐oxo‐trans‐2‐nonenal (ONE)‐protein adducts, research on the modification of a protein by (E,E)‐2,4‐decadienal is insufficient, and the chemical nature of (E,E)‐2,4‐decadienal‐protein adducts is not clear. This study may be useful for extending our understanding of the specific role of (E,E)‐2,4‐decadienal in the formation of modified proteins during the occurrence of chronic diseases and provides a theoretical reference for food quality and safety problems caused by protein carbonylation during processing and storage.

Ultrasonic pretreatment enhanced the glycation of ovotransferrin and improved its antibacterial activity

This study aims to evaluate the effect of ultrasonic pretreatment combined with glycation on the structural characteristics and antibacterial activity of ovotransferrin (OVT). Firstly, OVT (purity >90%) was isolated from egg white with a simple and efficient method. After the treatment of ultrasound and glycation, the browning degree of OVT increased with the rising power of ultrasound, while the number of free amino groups obviously decreased to 25.4%. Various spectrum detection showed that the structures of OVT have changed significantly, indicating the tertiary structure became more flexible and looser. The minimal inhibitory concentration of ultrasound glycated OVT were 25.0 and 32.1 μmol/L for E. coli and S. aureus, respectively. In summary, ultrasound-assisted glycation is an effective technique to improve the biological activity of OVT.

Assessing the effect of Maillard reaction with dextran on the techno-functional properties of collagen-based peptides obtained from bovine hides

The recovery of food processing co-products, in the meat sector, has become a hot topic. Based on previous studies, the enzymatic hydrolysis of bovine hides was proposed as a suitable and efficient recovery methodology to produce protein hydrolysates to be used in the food industry. It was found, however, that maximizing recovery yield lead to hydrolysates presenting very poor functional properties. Maillard reaction has been shown to modify the techno-functional properties of proteins without adding chemical agents. The glycation reaction occurred successfully as proved from the analysis of the free amino groups and the size exclusion chromatography (SEC). However, the glycated hydrolysates did not show an improvement in any of the techno-functional properties here assayed: foaming, gelling and emulsifying capacity. This lack of improvement was attributed to the low molecular weight of the peptides (less than 6.5 kDa in average, being the 60% of them lower than 3 kDa) required for recovering proteins from hides in high yields (>85%). When compared to non-hydrolysed collagen, the number of free amino groups per molecule in the hydrolysate is much lower, meaning that interactions between protein-protein and protein-matrix interactions are less evident.

Improvement of cross-linking and stability on cross-linked enzyme aggregate (CLEA)-xylanase by protein surface engineering

Abstract Cross-linked enzyme aggregate (CLEA) is a well-known enzyme immobilization technique that is efficient and cost-effective. In this study, we proposed a combination of the surface modification of an enzyme and CLEA to increase the number of free amino groups. Site-directed mutagenesis was applied to selected residues of xylanase from Aspergillus fumigatus RT-1 and the xylanase was subsequently cross-linked using glutaraldehyde. Surface analysis of the xylanase revealed that 9 residues were exposed to the environment and only 3 were lysines. Thus, four additional lysines were substituted for residues opposite of the catalytic region. After optimizing the CLEA parameters, a stable cross-linked mutant xylanase with the addition of BSA (mXyn-CLEA-BSA) was obtained where the enzyme was 1.09-fold, 1.35-fold and 1.77-fold more stable than the cross-linked recombinant xylanase with the addition of BSA (rXyn-CLEA-BSA), without the addition of BSA (rXyn-CLEA) and free enzyme (rXyn), respectively. In terms of reusability, rXyn-CLEA can be used up to 5 cycles, rXyn-CLEA-BSA and mXyn-CLEA up to 7 cycles and mXyn-CLEA-BSA up to 8 cycles until the total activity is lost. The increase in stability and reusability using this approach provides a promising biocatalyst that can be further utilized in the production of prebiotics in the biomass industry.

Study on structural, rheological and foaming properties of ovalbumin by ultrasound-assisted glycation with xylose.

This study was to evaluate the effect of ultrasound-assisted glycation with xylose on the foaming properties, physicochemical and structural characteristics of ovalbumin (OVA). The number of free amino groups in the glycated OVA (sOVA-X) significantly decreased with the increase of treatment time. The results obtained by circular dichroism (CD) and spectrofluorimetric measurements showed that there were slight changes on the subunits and secondary structure of OVA, indicating that the tertiary structure became more flexible and loose after the sonicated glycation treatment. The glycated OVA had higher solubility and foaming properties than the untreated samples. Therefore, ultrasound improved the glycation extent, and the changes in molecular structure were responsible for their different foaming ability and foaming stability. Our study also provided principle knowledge to understand how the viscosity and rheology were related to the foaming properties of OVA glycation by xylose. The results indicated that ultrasound-assisted glycation could be an excellent approach to improve the functional properties of OVA and promote its application in food industry.

Investigation of the properties of N-[(2-hydroxy-3-trimethylammonium) propyl] chloride chitosan derivatives

N-[(2-Hydroxy-3-Trimethylammonium) Propyl] Chitosan Chloride Derivatives (HTCC), based on low molecular weight crab chitosan, were synthesized by the alkylation reaction with a degree of substitution of 10–98%. The chemical structure was confirmed by 1H NMR and IR-spectra. Physical and chemical characteristics and a number of properties were defined. All HTCC derivatives were soluble at pH 7.4. HTCCs have an inhibitory capacity on the growth of the studied microorganisms. The introduction of quaternary ammonium groups into chitosan molecule contributed to the increase of antibacterial activity of derivatives. HTCC53 showed antifungal activity and at a concentration of 500 mg/ml completely inhibited the growth of mycelial fungi F. oxysporum, A. alternata and C. herbarum. When studying the ability of HTCCs to absorb DPPH radicals, it was found that samples of HTCC10 and HTCC40 showed high inhibitory capacity at a concentration of >15 mg/ml. It was shown that the chelating ability of HTCCs decreased by reducing the number of free amino groups. HTCC10-HTCC53 demonstrated the maximum values of chelating ability at a concentration of 4–10 mg/ml. Due to the solubility at neutral pH values and the properties shown, obtained chitosan derivatives can be used in clinical practice, pharmaceutical and food industries in the future.

Effect of ohmic heating on fundamental properties of protein in soybean milk

AbstractThe structure and functional properties of heated soybean milk protein were measured and compared between ohmic heating (17, 23, 30, and 37 V/cm) and common resistance wire heating. The results showed that the number of free amino groups increased by 14%; total sulfhydryl content and surface hydrophobicity decreased; the protein structure was not significantly changed; functional properties and foaming activity were reduced by 10, 15, 35, and 40% for the different voltage ohmic heating; foam stability was reduced by 28.3, 12.9, 18.3, and 8.3%, respectively; the EAI of ohmic heating increased by 38%, whereas the ESI decreased by 65%. Soybean protein has been reported to be denaturalized in traditional heating. As a resistor, in situ vibration was conducted inside the molecule when it is heated via electricity. It can be assumed that ohmic heating is conducive to the improvement of functional properties, digestion, and absorption of soybean milk protein.Practical applicationsThis study explored effects of voltage ranges on soybean milk quality and structure and functional properties. After ohmic heating treatment, the structure and functional properties of protein in soybean milk greatly changed relative to traditional heating treatment. The application of ohmic heating for soybean milk was found very successful in reducing heating time and producing nutrient products. The same goes for the other fruit and vegetable products, it is of great directive significance to ohmic heating processing industry for them.

Reactive Landing of Gramicidin S and Ubiquitin Ions onto Activated Self-Assembled Monolayer Surfaces.

Using mass-selected ion deposition combined with in situ infrared reflection absorption spectroscopy (IRRAS), we examined the reactive landing of gramicidin S and ubiquitin ions onto activated self-assembled monolayer (SAM) surfaces terminated with N-hydroxysuccinimidyl ester (NHS-SAM) and acyl fluoride (COF-SAM) groups. Doubly protonated gramicidin S, [GS + 2H]2+, and two charge states of ubiquitin, [U + 5H]5+ and [U + 13H]13+, were used as model systems, allowing us to explore the effect of the number of free amino groups and the secondary structure on the efficiency of covalent bond formation between the projectile ion and the surface. For all projectile ions, ion deposition resulted in the depletion of IRRAS bands corresponding to the terminal groups on the SAM and the appearance of several new bands not associated with the deposited species. These new bands were assigned to the C=O stretching vibrations of COOH and COO- groups formed on the surface as a result of ion deposition. The presence of these bands was attributed to an alternative reactive landing pathway that competes with covalent bond formation. This pathway with similar yields for both gramicidin S and ubiquitin ions is analogous to the hydrolysis of the NHS ester bond in solution. The covalent bond formation efficiency increased linearly with the number of free amino groups and was found to be lower for the more compact conformation of ubiquitin compared with the fully unfolded conformation. This observation was attributed to the limited availability of amino groups on the surface of the folded conformation. Our results have provided new insights on the efficiency and mechanism of reactive landing of peptides and proteins onto activated SAMs. Graphical Abstract ᅟ.

Preparation and Biophysical Characterization of Poly(amidoamine) Dendrimer-Poly(acrylic acid) Graft.

A series of PAMAM dendrimer generation 5-poly(acrylic acid) grafts were prepared to evaluate the potential use of dendritic grafts as a drug encapsulated nanocarrier. The structural features of the synthesized polymer graft were identified by FT-IR and 1H-NMR spectra and the biophysical properties were characterized by measuring its particle size and zeta potential. The prepared dendrimer G5-PAA grafts had particle size in the range of 600 to 900 nm and the size increased proportionally with the number of PAA on dendrimer surface. The electrostatic property of the dendrimer G5-PAA, carried out by HPLC reversed phase column analysis and the measurement of zeta potential, revealed that both migration time and zeta potential were dependent on the number of grafted PAA. The number of free amino groups on dendrimer G5-PAA, determined quantitatively by fluorescamine assay, was in a reverse order with the reaction mole ratio of dendrimer to PAA. In addition, dendrimer G5-PAA showed a pH-dependent solubility in aqueous solution with characteristic pH region of solubility, depending on the dendrimer generation. The observed biophysical properties indicate that PAMAM dendrimer G5-PAA is promising as a drug encapsulated nanocarrier.

Immobilization of horseradish peroxidase as crosslinked enzyme aggregates (CLEAs)

Herein we report the successful preparation of catalytically active crosslinked enzyme aggregates (CLEAs®) of peroxidase (HRP, EC 1.11.1.7) from horseradish roots (Armoracia rusticana or Cochlearia armoracia) using various aggregation agents, followed by crosslinking with glutaraldehyde. The percent activity recovery of the HRP-CLEAs was 83% of that of the native enzyme. The CLEAs were prepared in the presence of egg albumin (37mg/mL) and pentaethylenehexamine (PEHA; 10mM). The albumin played a significant role in stabilizing the CLEA particles, and the PEHA was necessary to obtain fully crosslinked HRP aggregates. Because HRP possesses only six Lys (lysine) residues, the addition of PEHA increases the number of free amino groups (–NH2) on the outer surface of the enzyme, thus facilitating the crosslinking. A potential application of HRP aggregates is envisaged for coupled oxidation reactions with cholesterol oxidase in the biosensor field.

Effects of transglutaminase on the rheological and Mixolab thermomechanical characteristics of oat dough

The effects of added transglutaminase (TG) on the rheological and thermal properties of oat dough were evaluated. Mixolab, rheometer, and differential scanning calorimetry (DSC) were used to analyse the oat dough for changes in thermomechanical, rheological, and thermal properties. TG had distinct effects on dough water absorption, modified viscoelastic behaviour, and enhanced thermal stability. The dough also exhibited a decrease in the number of free amino groups after TG treatment, confirming protein cross-linking catalysed by TG. Electrophoresis of TG-treated oat protein fractions using SDS–PAGE, which was also used to analyse the effects of TG on the protein fractions of oat flour; it showed that both globulin and avenin were good substrates for TG.

Immobilization of Modified Papain with Anhydride Groups on Activated Cotton Fabric

Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method. Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the cotton fabric immobilized modified papain has potential applications in the functional textiles field.

Preparation and Biophysical Characterization of Pluronic F127-Dendrimer Conjugate as a Delivery Agent of Antisense Oligonucleotides

A series of pluronic F127 conjugated PAMAM dendrimer generation 5 were prepared to evaluate the potential use of dendrimer-pluronic conjugates as a delivery agent of antisense oligonucleotides. The structural features of the conjugates were identified by FT-IR and 1H-NMR spectra. The prepared dendrimer generation 5-pluronic F127 conjugates showed particle size in the range of 50 to 130 nm with reduced zeta potential. The electrophoretic property of conjugates was further confirmed by capillary zone electrophoresis (CZE), demonstrating the decrease in electrophoretic mobility of conjugates as the number of pluronic F127 on surface of dendrimer increases. The number of free amino groups on a pluronic F127 conjugated dendrimer was quantitatively determined by fluorescamine assay, measuring the fluorescence intensity of amino groups reactive to fluorescamine on dendrimer generation 5-pluronic F127 conjugates. In MTT assay, dendrimer G5-pluronic F127 conjugates were safe on gingival fibroblast cells and acute toxicity was reduced as the mole ratio of dendrimer to pluronic increased. Complex formation of dendrimer G5-pluronic F127 conjugates with oligonucleotides was dose dependent.

Crosslinking of sodium caseinate by a microbial transglutaminase.

Sodium caseinate is an effective substrate for transglutaminase. Crosslinking takes place at fast reaction rates resulting in high degrees of crosslinking. The polymers can be hydrolysed by trypsin, but the proteolysates contain residual portions of non- or partlyhydrolysed aggregates. Crosslinking of hydrolysed sodium caseinate decreased the number of free amino groups, but leads only to a very small increase in molecular weight of the peptides.

Doxorubicin-loaded gelatin nanoparticles stabilized by glutaraldehyde: Involvement of the drug in the cross-linking process

The possible involvement of the primary amino group of doxorubicin (DXR) in the cross-linking process of gelatin nanoparticles stabilized by glutaraldehyde was investigated. Nanoparticles were characterized with regard to particle size, drug content, enzymatic degradation and cross-linking degree. The size of nanoparticles was around 100–200 nm and DXR was loaded with an entrapment efficiency of 42%. Upon the study of crosslinking rate, DXR-loaded nanoparticles showed a greater number of free amino groups than the unloaded ones. This should be due to a competition between the amino group of DXR and the amino groups of the gelatin chains during the cross-linking process. Hence, a binding of a drug fraction to the protein matrix via glutaraldehyde was hypothesized and confirmed by the results of a thin-layer chromatography (TLC) analysis. According to the in vitro study only a little fraction of DXR was released as free drug (8%) when the nanoparticles were put in saline solution. The addition of proteolytic enzymes disrupts the matrix structure producing the release of a further 10–15% of the drug loading which was entrapped in the nanoparticle matrix. The remaining part of the drug corresponds to DXR covalently linked to peptide residues produced by nanoparticle digestion.

An immunological approach to monitor protein lactosylation of heated food model systems

Poly-L-lysine, a high molecular weight synthetic compound with a large number of free amino groups, was glycated with lactose through the Maillard reaction (MR). Polyclonal mouse and rabbit antibodies (Abs) were obtained and characterised by competitive ELISA (enzyme-linked immuno assay), immunoblotting and cytofluorimetric assays. Results demonstrated that the Abs react specifically with lactosylated food proteins, i.e. bovine serum albumin (BSA) and casein and do not cross react with the underivatised proteins. The immunoreactivity between Abs and lactose-BSA adducts was strongly inhibited by N-ϵ-deoxy-lactulosyl-L-lysine, indicating that this typical MR product is recognised specifically by our Abs. These reagents can be a useful tool to monitor the MR reaction between carbohydrates and lysine, which occurs during the thermal treatment of food.