Determination Of Free Amino Groups Research Articles

Reduction of maillard browning in spray dried low-lactose milk powders due to protein polysaccharide interactions

Lactose hydrolysed concentrated milk was prepared using β-galactosidase enzyme (4.76U/mL) with a reaction period of 12 h at 4 °C. Addition of polysaccharides (5 % maltodextrin/β-cyclodextrin) to concentrated milk either before or after lactose hydrolysis did not result in significant differences (p > 0.05) in degree of hydrolysis (% DH) of lactose and residual lactose content (%). Three different inlet temperatures (165 °C, 175 °C and 185 °C) were used for the preparation of powders which were later characterised based on physico-chemical and maillard browning characteristics. Moisture content, solubility and available lysine content of the powders decreased significantly, whereas, browning parameters i.e., browning index, 5-hydroxymethylfurfural, furosine content increased significantly (p < 0.05) with an increase in inlet air temperature. The powder was finally prepared with 5 % polysaccharide and an inlet air temperature of 185 °C which reduced maillard browning. Protein-polysaccharide interactions were identified using Fourier Transform infrared spectroscopy, fluorescence spectroscopy and determination of free amino groups in the powder samples. Maltodextrin and β-cyclodextrin containing powder samples exhibited lower free amino groups and higher degree of graft value as compared to control sample which indicated protein-polysaccharide interactions. Results obtained from Fourier Transform infrared spectroscopy also confirmed strong protein-polysaccharide interactions, moreover a significant decrease in fluorescence intensity was also observed in the powder samples. These interactions between the proteins and polysaccharides reduced the maillard browning in powders.

Cytotoxic, antioxidant, and antiglycation activities, and tyrosinase inhibition using silver nanoparticles synthesized by leaf extract of Solanum aculeatissimum Jacq

ABSTRACT The present study aimed to determine the biological properties of an extract of Solanum aculeatissimum aqueous extract (SaCE) alone as well as silver nanoparticles (AgNPs) generated by green synthesis utilizing S. aculeatissimum aqueous extract (SaCE). These synthesized SaCE AgNPs were characterized using UV-VIS spectrophotometry, scanning transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), zeta potential (ZP), dynamic light scattering (DLS). Determination of total polyphenols, flavonoids, saponins content was conducted. In addition, high performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to identify constituents in this extract. Antioxidant activity was determined by DPPH radical scavenging and ferric ion reducing power (FRAP) methods. Antiglycation activity was demonstrated through relative mobility in electrophoresis (RME) and determination of free amino groups. The inhibitory activity on tyrosinase was also examined. Molecular docking analyses were performed to assess the molecular interactions with DNA and tyrosinase. The antitumor activity SaCE was also measured. Phytochemical analysis of SaCE and AgNPs showed presence polyphenols (1000.41 and 293.37 mg gallic acid equivalent/g), flavonoids (954.87 and 479.87 mg rutin equivalent/g), saponins (37.89 and 23.01% total saponins), in particular steroidal saponins (aculeatiside A and B). Both SaCE and AgNPs exhibited significant antioxidant (respectively, 73.97%, 56.27% in DPPH test, 874.67 and 837.67 μM Trolox Equivalent/g in FRAP test) and antiglycation activities (72.81 and 67.98% free amino groups, results observed in RME). SaCE and AgNPs presented 33.2, 36.1% inhibitory activity on tyrosinase, respectively. In silico assay demonstrated interaction between steroidal saponins, DNA or tyrosinase. SaCE exhibited antitumor action against various human tumor cells. Data demonstrated that extracts SaCE alone and AgNPs synthesized from SaCE presented biological properties of interest for application in new therapeutic formulations in medicine.

The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion

Abstract A two-stage in vitro gastrointestinal (GI) digestion model was used to assess the survivability of antioxidant peptide fractions with different molecular weights (MW). Research on MW distribution showed that peptides above 3000 Da were more easily digested by gastric digestion than those below 3000 Da. And peptides tended to be more hydrolyzed by intestinal digestion than by gastric digestion. Determination of free amino groups suggested that peptide bonds in the peptides above 3000 Da were more inclined to be cut off during the GI digestion. However, the amount of free amino acid (AA) increased as the MW decreased and accounted for 30%–40% (w/w) of the GI digests. The peptides below 1000 Da exhibited the best initial and surviving antioxidant activities in 2, 2′-azinobis (3-ethylbenzothiszoline-6-sulphonic acid) diammonium salt (ABTS + ), hydroxyl radical scavenging and ORAC value. In conclusion, casein-derived antioxidant peptides below 1000 Da were a promising antioxidant administered orally. Industrial revelance: Numerous products are already on the market or under development by food companies, exploiting the potential of food-derived bioactive peptides. To exert physiological effects in vivo, bioactive peptides must survive the gastrointestinal (GI) digestion, and then reach their target sites after absorption. So far, only a few among the numerous bioactive peptides displaying activities in vitro have been proven effective in vivo. The real bioavailability of quite an amount of marketed products containing bioactive peptides is unclear. However, our research conducted further examination of bio-accessibility of casein-derived antioxidant peptides and assessed the effect of MW on the survivability of casein-derived antioxidant peptides after the simulated GI digestion. The survivability of peptides was above 60% after the GI digestion. And the casein-derived peptides below 1000 Da exhibited the best initial and surviving antioxidant activities. The enriched antioxidant peptide fraction above could be a promising candidate for dietary supplements and nutraceuticals with good GI survivability and bio-accessibility.

Comparative study of gelation and cross-link formation during enzymatic texturisation of leguminous proteins

Abstract Isopeptide bonds that resulted from protein cross-linking, catalysed by a microbial transglutaminase (MTG), substantially contributed to the physicochemical modification of leguminous proteins. For the development of texturised vegetable protein (TVP)-based foodstuffs using MTG, valid methods for an efficient control of the gelation process are a prerequisite. Formation of e-(γ-glutamyl)lysine cross-links in a simple food model system, containing proteins from soy [Glycine max (L.) Merr.] or pea (Pisum sativum L.), was monitored by quantification of the e-(γ-glutamyl)lysine bond via HPLC-MS and by determining the depletion of free amino groups during cross-linking spectrophotometrically after derivatisation with o-phthaldialdehyde (OPA). Increasing gel strengths during incubation with MTG were measured via texture analysis. The OPA method proved too unspecific for controlling the enzymatic gelation process of leguminous proteins. Specifically for each substrate, the levels of isopeptide cross-links, detected via HPLC-MS analysis, correlated well with the gel strength of the texturised proteins (R2 = 0.961–0.994). Rapidly measurable, gel strength was shown to be a reliable command variable for managing MTG-induced gelation. Its use also allowed indirect estimation of the degree of feasible cross-linking. Industrial relevance Leguminous proteins represent a valuable alternative to animal proteins for the manufacture of texturised foodstuffs. However, due to the poor gelling properties of the native proteins, their potential is still unexploited. For the development of TVP using MTG, simple gel strength measurements were shown to be a valid tool for the control of the gelation process. For this purpose, unlike OPA determination of free amino groups and LC-MS analysis of isopeptide bonds, tedious sample preparation is not required.

Free amino group content of serum albumin microcapsules. III. A study at low pH values

Microcapsules were prepared by interfacial cross-linking of human serum albumin (HSA) with terephthaloyl chloride (TC). Determination of free amino groups was performed on lyophilized microcapsules with trinitrobenzene-sulfonic acid (TNBS). In two series of assays conducted with phosphate buffers, pH 7.4 and 8, respectively, microcapsules exhibited a high −NH 2 content (> 330 μmol/g dry weight), with only a slight decrease when raising TC concentration from 2.5 to 5% TC (w/v) and/or reaction time from 30 to 60 min. When the phosphate buffer, pH 7.4 (ionic strength (μ): 0.40), was replaced by an acetate buffer, pH 7.4 (μ: 0.66), supplemented with 0.125 or 0.25 M CaCl 2, microcapsule −NH 2 content was reduced from 346 to 134 and 168 μmol/g, respectively. Control batches prepared with the acetate buffer without adding CaCI 2 also had a reduced −NH 2 content (182, μmol/g), while a higher value (302 μmol/g) was found for microcapsules prepared using a phosphate buffer with a 0.66 ionic strength. Finally, a sharp decrease in −NH 2, content was also found for microcapsules prepared from acetate buffers at pH 5.9 (174 μmol/g) and 6.8 (158 μmol/g), as compared with microcapsules prepared from phosphate buffers at the same pH values (438 and 388 μmol/g, respectively).

Measurement of proteolysis in cheese: relationship between phosphotungstic acid-soluble N fraction by Kjeldahl and 2,4,6- trinitrobenzenesulphonic acid-reactive groups in water-soluble N

Free amino groups produced during cheese ripening are used to indicate the extent of cheese proteolysis. Several studies have shown a high correlation between the level of free amino acids and the flavour of Gouda (Aston et al. 1983) or Comté (Grappin &amp; Berdagué, 1989). Measurement of the level of free amino acids seems useful for the investigation of flavour chemistry in cheese (Lemieux et al. 1990). The determination of N fractions is often used to estimate the degree of proteolysis in cheese, but since this procedure is laborious and time consuming several attempts have been made to replace it by more rapid methods (Ardö &amp; Meisel, 1991). Since its introduction by Satake et al. (1960), the 2,4,6-trinitrobenzenesulphonic acid (TNBS) method has been widely used for the determination of free amino groups. Because TNBS does not react with the imino groups of histidine and proline or the hydroxyl groups of tyrosine, serine or threonine, it has been accepted as a selective reagent for primary amino groups (Burger, 1974). Measurement of N by Kjeldahl in the phosphotungstic acid (PTA)–sulphuric acid extract (Gripon et al. 1975) estimates the N of free amino acids and low molecular mass peptides. The purpose of this study was to compare the TNBS and PTA-soluble N methods in order to find out whether the TNBS procedure can replace that of PTA-soluble N in the determination of a cheese proteolysis index.

Partitioning of chemically modified low-density lipoprotein in aqueous polymer two-phase systems

Aqueous polyethylene glycol (PEG)-dextran two-phase systems containing 10 m M Tris·HCl (pH 7.4) were used for the partitioning of chemically modified low-density lipoprotein (LDL). Anionic modification connected with an increase in the negative surface charge of lipoproteins favours the accumulation of modified LDL in the top phase. The partition coefficient increases depending on the extent of modification. Cationic modification yields lower values for the partition coefficient. Positively charged LDL favours a bottom-phase accumulation. With weakly charged and nearly neutral particles, the Van der Waals interaction between polymer and particle preponderates over electrostatic interactions, leading to a favoured accumulation of LDL in the PEG-rich top phase. Results of measurements of the relative electrophoretic mobility and the determination of free amino groups are in agreement with the calculated values of the partition coefficient. Because the partitioning of LDL is accompanied by aggregation at the interface, experimental techniques have to be carefully standardized. Subtle differences in the surface properties of modified LDL can be detected.

Conformational changes and physicochemical properties of transferrin upon derivatization with cholesterol

Transferrin was chemically modified by covalent linkage of cholesteryloxycarbonyl-6-aminohexanoic acid to the lysine amino groups. Cholesteryloxycarbonyl-6-aminohexanoic acid was prepared from cholesteryl chlorocarbonate and 6-aminohexanoic acid. This derivative was attached by an amide bond to the free amino groups of transferrin. The level of derivatization was quantified by amino acid analysis and determination of free amino groups by the trinitrobenzene sulfonic acid method. The physicochemical characteristics of native and derivatized transferrin were studied by several methods. Surface activity was determined by the pressure increases at the air/water interface. Surface hydrophobicity was checked by anilino naphthalene sulfonic acid titration. Lipid-protein interactions were determined by intrinsic fluorescence changes, with the bilayers in the fluid and gel state. The results obtained show that derivatization renders the protein highly hydrophobic. Nevertheless, the surface activity or the interaction with lipids do not correlate with the hydrophobicity of the molecule, thus suggesting the formation of some type of internal micelles or a strongly packed structure.

Determination of free amino group content of serum albumin microcapsules: II. Effect of variations in reaction time and in terephthaloyl chloride concentration

Microcapsules were prepared through interfacial cross-linking of human serum albumin (HSA) using terephthaloyl chloride (TC) at a constant pH of 9.8. Variations were performed in reaction time (for a 2.5% w/v TC concentration) and in TC concentration (for a 30 min reaction time). Determination of free amino groups was performed on lyophilized microcapsules by means of a back titration method using trinitrobenzenesulfonic acid (TNBS). When prolonging the reaction time from 2 to 30 min. microcapsule-NH 2 content progressively decreased from 154 to 60 μmol/g dry weight. These results were in good agreement with those of a previous study conducted using Fourier transform infrared spectroscopy. A slight recovery of free-NH 2 was observed after 60 and 120 min. Increasing TC concentration from 1 to 5% (w/v) resulted in a progressive decrease in microcapsule-NH 2 content from 99 to 38 μmol/g.

Determination of free amino group content of serum albumin microcapsules using trinitrobenzenesulfonic acid: effect of variations in polycondensation pH

Microcapsules were prepared from human serum albumin (HSA) through an interfacial cross-linking process using terephthaloyl chloride at various pH values (5.9–11). Determination of free amino groups was performed on lyophilized microcapsules by means of a back titration method using trinitrobenzenesulfonic acid (TNBS). Increasing reaction pH was shown to result in a progressive decrease of microcapsule free amino groups. Moreover, two groups of microcapsules could be distinguished with respect to their -NH 2 content: those obtained at low pH values (8 and < 8) which contained more than 400 μmol g dry weight, and those prepared from pH 9, whose -NH 2 content did not exceed 110 μmol g dry weight. These results were compared with previous findings concerning the involvement of hydroxy and carboxy groups of HSA microcapsules as a function of pH, that had been obtained from FT-IR spectroscopic studies.

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986)] J. Biochem. Biophys. Methods 12, 349–354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5′- O-(4, 4′-dimethoxytrityl)-thymidine-3′- O-(2, 4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4, 4′-dimethoxytrityl cation into the solution. The released 4, 4′-dimethoxytrityl cation, which has a strong absorption at 498 nm ( ε 498 = 70 000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4, 4′-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.

4,4′-Dimethyloxytrityl chloride: a reagent for the spectrophotometric determination of polymer-supported amino groups

A rapid and sensitive spectrophotometric method for the determination of free amino groups on a solid support is described. The method is based on the reaction of polymer-supported amino groups with an excess of 4,4′-dimethoxytrityl chloride (DMTrCl) in the presence of tetrabutylammonium nitrate as a catalyst. The solid support was washed thoroughly to remove the excess of reagent and subsequently treated with perchloric acid solution to release the DMTr cation. The DMTr cation was then determined spectrophoto-metrically at 498 nm (ε498= 70 000 l mol–1 cm–1). The method was found to be useful for the determination of solid-supported amino groups and for monitoring the functionalisation of polymer supports used for the synthesis of peptides and oligonucleotides. In addition the polymer supports can be reused after the determination of the solid-supported amino groups.

Gossypol prevents activation of purified proenzyme of human seminal plasma acidic proteinase

Gossypol inhibits the potential activity of the proenzyme form of human seminal plasma acidic proteinase, but has no effect on the active enzyme under the conditions tested. Inhibition of proenzyme is rapid and pH-dependent: 50% inhibition can be observed at gossypol concentrations of approx. 1.5 · 10 −5 M. SDS-polyacrylamide gel electrophoresis indicates that treatment of proenzyme with gossypol prevents the formation of active enzyme that normally occurs on acidification. Determination of free amino groups with 1-fluoro-2,4-dinitrobenzene suggests that gossypol reacts with 7.8 out of the 11.0 lysine residues in proenzyme: such a reaction could interfere with the activation process.

A simple method for the estimation of amino groups on insoluble matrix beads

A method for the determination of free amino groups on solid supports is proposed. The method is based on the reaction of the matrix with excess 2,4,6-trinitrobenzenesulfonic acid (TNBS) and subsequent quantitative determination of unreacted TNBS by reaction with glycine. The method is simple and reproducible and does not require the solubilization of the matrix, therefore proving suitable even for crosslinked insoluble supports.

Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction

A rapid and sensitive method for the quantitative determination of free amino groups during solid-phase peptide synthesis has been developed. The technique involves the reaction of the free amine with ninhydin under carefully controlled conditions and the determination of the resulting chromophore in solution at 570 nm. All of the blue chromophore of the ninhydrin reaction with primary amines is found in solution and the beads of the polystyrene support are colorless. An ion-exchange mechanism for the formation of blue beads by previous methods is proposed. The technique is useful for measuring the total number of peptide chains on the resin and, especially, for monitoring the progress of the coupling reaction. It is possible to determine when the formation of a peptide bond is 99.9% complete.

New Method for the Determination of Free Amino Groups in Intact Pure Proteins: Relationship to Available Lysine

A new rapid method for the quantitative and routine determination of free amino groups in intact pure proteins has been developed. Primary amino groups are labeled with fluorescamine and the labeled groups are detected by absorption spectroscopy in the range 375-390 nm. The amino group concentration can be determined in a few minutes without hydrolyzing the labeled protein and extracting a lysine derivative. The method was tested with the following proteins: lysozyme, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, ribonuclease, ribonuclease-S-peptide, and alphasl-casein B. Application of this method to the estimation of available lysine is discussed.

Automated Determination of Free Amino Groups in Serum and Plasma Using 2,4,6-Trinitrobenzene Sulfonate

Abstract A simple automated method is described for determining the level of total free amino acids in the blood. The method utilizes the AutoAnalyzer, and is based on the formation of colored complexes by uniting free amino groups with 2,4,6-trinitrobenzene sulfonate (TNBS). Proteins do not interfere because the free amino acids are first separated by dialysis. Characteristics of the reaction and potential clinical applications of the procedure are discussed.

Determination of free amino groups in proteins by trinitrobenzenesulfonic acid

A sensitive method to determine the free amino groups in proteins is presented that makes use of the reagent 2,4,6-trinitrobenzenesulfonic acid. The relationship between absorbance and concentration (up to 1 mg/ml) was linear and the color was developed under more gentle conditions than with the ninhydrin method. It was found that sodium dodecyl sulfate was bound to some of the ϵ-amino groups of lysine in bovine serum albumin, ovalbumin, and human γ-globulin and rendered the involved amino groups unreactive toward TNBS. Partial reaction of 4 M potassium thiocyanate at 40°C for 2 hr with the free amino groups in bovine serum albumin, ovalbumin, human γ-globulin, and lactalysate was demonstrated by this method. The method was useful to study the reaction of the free amino groups of bovine serum albumin with formaldehyde.

Nitrosation of Soil Organic Matter: I. Nature of Gases Evolved During Nitrous Acid Treatment of Lignins and Humic Substances

AbstractManometric and infrared studies of the gaseous products formed by reacting HNO2 with humic and fulvic acids, lignins. lignin‐building units, and polyphenols showed the presence of N2, N2O, and CO2. In addition, methyl nitrite (CH3ONO) occurred in the gases obtained from lignins and certain phenolic derivatives. The CH3ONO originated through dismutation of phenolic ethers; the CO2 from decarboxylation of aromatic acids and cleavage of ring structures. Aromatic‐like substances interfere with the determination of free amino groups in soil organic matter by the Van Slyke nitrous acid method.

PREPARATION OF AN ESTRONE–PROTEIN CONJUGATE

A procedure is described for the preparation of an estrone–protein conjugate. The 17-amino derivative of estrone was prepared and converted by phosgenation to estrone-17-isocyanate. The latter was coupled to rabbit serum albumin. Ultraviolet analysis and determination of free amino groups of both albumin and estrone–albumin conjugate showed that the latter contained about 25 estrone residues per albumin molecule. Electrophoretic analysis provided additional evidence that coupling had occurred at the ε-amino groups of the lysine residues of albumin.