Determination Of Amino Acid Composition Research Articles

Amino Acid Composition Determination From the Fractional Mass of Peptides

ABSTRACTA peptide's fractional mass is directly associated with its elemental composition and thus amino acid composition. Here it is demonstrated that a peptide's fractional mass alone can be a useful identifier or indicator of that composition for small to mid‐sized peptides (5–7 amino acids) and can significantly reduce the number of viable amino acid compositions for larger peptides (> 8 residues) to include or exclude certain possibilities. Separate consideration of the integer portion of the peptide's mass helps to reduce the number of possibilities where many duplicate fractional mass values are found. Adoption of this fractional mass strategy should aid approaches that are presently employed for peptide identification, including in the use of mass map data to search protein databases for proteomics applications.

Exploring protein derivative profiles in cheese whey through native Candida tropicalis fermentation

ABSTRACT Native yeasts produce most peptides and amino acids from cheese whey fermentation. The research aimed to determine the protein, peptides, and amino acid profiles regarding whey fermentation by spontaneous fermentation and re-inoculation by native yeasts as a basis for determining potential yeasts in amino acid production. The selected native yeasts were identified through genome-based analysis on the DNA base sequence of the Internal Transcribed Spacer (ITS) region. The molecular identification shows that isolate CT1 is 95.62% comparable to Candida tropicalis strain VIT-NN02, CT2 is 98.85% same to stain NB5Y, and CT3 is 99.42% similar to GT 6. Whey fermentation was conducted by inoculating 3% each native C. tropicalis and without inoculation for spontaneous fermentation, then incubated for 48 hours at ambient temperature (26 ± 2°C). Unlike the other samples, the spontaneously fermented mozzarella whey was incubated without inoculation after cheese separation. After 48 hours, fermented whey was kept at 4°C for examination. The Kjeldahl method evaluated protein concentration, Folin-Ciocalteau determined peptide levels, and HPLC determined amino acid composition. C.tropicalis was identified as cheese whey native yeasts, and all treatments decreased protein and peptides while increased the total amino acid includes every single amino acid value except histidine during isolate CT1 whey fermentation. Candida tropicalis CT2 shown as potential isolate for the amino acid production from whey, with the protein contents of 0.846 ± 0.151%, protein decreases of 0.384 ± 0.186, peptide levels of 264.75 ± 40.40ppm, peptide decreases of 99.87 ± 37.96 and the highest total amino acid of 6149.56ppm.

Effect of white jade snail secretion on antioxidant capacity and intestinal microbial diversity in mouse model of acute gastric ulcer.

In the present work, acute gastric ulcer models were constructed by administering hydrochloric acid/ethanol. The mice ingested white jade snail secretion (WJSS) through gastric infusion. Ulcer areas in gastric tissue were recorded, and malondialdehyde (MDA) and superoxide dismutase (SOD) were also measured. Notably, high-throughput 16S rDNA analysis of intestinal flora and determination of amino acid composition in feces were performed to understand the effect of WJSS on model mice. Compared with the control group, the ulcer area in the WJSS low-, medium- and high-concentration groups declined by 28.02%, 39.57% and 77.85%, respectively. MDA content decreased by 24.71%, 49.58% and 64.25%, and SOD relative enzyme activity fell by 28.19%, 43.37% and 9.60%, respectively. The amounts of amino acids in the low-, medium- and high-concentration groups were slightly lower, and probiotic bacteria such as Bacteroidetes and Lactobacillales increased in different-concentration WJSS groups. Adding WJSS contributes to the establishment of beneficial intestinal flora and the absorption of amino acids. Our results showed that WJSS has a beneficial effect on inhibiting hydrochloric acid-ethanolic gastric ulcers, suggesting that WJSS has excellent potential as a novel anti-ulcer agent. Combined with ulcer area, MDA content, SOD content, gut probiotics and other indicators, a high concentration of WJSS had the best protective effect on acute gastric ulcer. © 2023 Society of Chemical Industry.

An Accurate Estimate of the Amino Acid Content of Human Milk Collected from Chinese Women Adjusted for Differences in Amino Acid Digestibility

BackgroundThe amino acid (AA) composition of human milk is used to define the AA requirements of the infant. Thus, it is important that estimates of composition be as complete and accurate as possible. When determining AA composition using standard hydrolysis methods, some AAs are progressively destroyed while others are incompletely released. For accuracy, AA composition needs to be determined using multiple hydrolysis times. The true ileal digestibility of AAs also needs to be taken into consideration. ObjectiveThe objective was to bring together AA compositional (determined using multiple hydrolysis intervals) and digestibility data determined using the piglet to give an estimate of the absorbed AA profile of human milk with reference in particular to Asian females. MethodsMature milk was collected from Chinese females. AA analysis using multiple hydrolysis intervals and a nonlinear regression model was used to accurately estimate AA composition. Human milk, as well as a protein-free diet, were fed to piglets (n = 6), and ileal digesta were collected (piglet age, 21 d) to determine the true ileal AA digestibility of AAs in human milk. ResultsTrue ileal AA digestibility coefficients ranged from (mean ± standard error of the mean) 0.61 ± 0.081 for tyrosine to 1.01 ± 0.030 for tryptophan, with a digestibility for total nitrogen of 0.90 ± 0.013. Convergence criteria were met for the modeling for each AA, and the model had a level of significance of P < 0.0001 for each AA. The amount of available AAs (total AA content as per the model prediction multiplied by the true ileal AA digestibility coefficient determined in the piglet) are reported. ConclusionsAn estimate of the absorbed AA profile of mature milk collected from Chinese females is provided. For the first time, data is presented for cysteine.

Studies on latent and soluble polyphenol oxidase from Moringa oleifera Lam. leaves

Polyphenol oxidase (PPO) from Moringa oleifera leaves was investigated to unravel its unique properties that can be exploited for possible biotechnological application(s).Soluble and latent PPO were extracted and purified to homogeneity using a combination of three-phase partitioning and gel filtration chromatography. The purity and molecular weight of the purified protein were estimated by SDS-PAGE and further by nano liquid chromatography-electrospray ionization tandem mass spectrometry (Nano-LC-ESI-MS/MS) and amino acid composition determination. Physicochemical and kinetic properties of the purified Moringa oleifera PPO (moPPO) were investigated following established protocols.Two soluble isoforms and a latent form of PPO were purified from the leaves of Moringa oleifera. All purified proteins were monomeric with molecular weights ranging from 10 to 65 kDa. PPO with the lowest molecular weight was further confirmed to be 7.6 kDa, 10.5 ± 0.06 kDa 7.2 kDa, and 8.61 ± 1.69 kDa as estimated by Nano-LC-ESI-MS/MS, SDS-PAGE, amino acid composition determination, and SEC respectively. The soluble proteins have pH optima at pH 6.5 and slightly basic pH 8.0, and that of the latent PPO was found to be 5.5 and 8.0 respectively. The optimal temperature for the purified enzymes was between 15 ᵒC – 25ᵒC and their catalytic efficiency was between 0.4 - 1.9 x 105 M−1s−1.The study concluded that M. oleifera leaves contain at least three forms of PPO, a novel low molecular weight peptide enzyme, a soluble form and a latent form with high kcat/Km similar to those obtainable from other sources which could be of interest in biotechnological applications.

Study of Epilobium angustifolium L. amino acids content by HPLC method

The use of plant raw materials is one of the areas of modern pharmaceutical science in the production of herbal drugs. The genus Epilobium counts more than 200 species, many species of which are used in traditional medicine. Among the Epilobium species, Epilobium angustifolium is one of the well-known medicinal plants which have been used worldwide in habitual medicine. There is insufficient information in the literature on the biologically active substances of Epilobium angustifolium L. The presence of three major polyphenol groups: phenolic acids, flavonoids, and ellagitannins were identified in E. angustifolium extracts. Traditionally, the infusion of leaves of this plant could be useful for headaches, cold and gastrointestinal disorder. The Epilobium angustifolium L. as an insufficiently studied plant is a promising object of study, including amino acids composition. To assess the relationship between the production of primary metabolites and their possible therapeutic properties, we analyzed the amino acid profile of the plant Epilobium angustifolium used in traditional medicine. The study of compounds generated by plants as a result of defense mechanisms permits an understanding of the molecular mechanism involved in their medicinal properties.&#x0D; The aim. Thus, the aim of the study was to conduct an HPLC analysis of the amino acids of E. angustifolium to establish the prospects for the use of the raw materials in medical and pharmaceutical practice. The results of the current study will be used in further breeding programs aimed to obtain an industrial form of E. angustifolium suitable for pharmaceutical and food applications.&#x0D; Materials and methods. The determination of amino acids composition of Epilobium angustifolium was conducted using Agilent 1200 (Agilent Technologies, USA).&#x0D; Results. The HPLC method identified sixteen free amino acids and seventeen bound amino acids in the Epilobium angustifolium herb. The studies have shown that Epilobium angustifolium L. herb is mainly composed of free amino acids such as L-phenylalanine (1.65 µg/mg), L-glutamic acid (1.51 µg/mg), L-arginine (1.24 µg/mg), L-alanine (0.98 µg/mg) and L-aspartic acid (0.57 µg/mg), which were presents in the greatest amount. The dominant bound amino acids in the studied raw material were L-glutamic acid, L-aspartic acid, L-leucine, and L-alanine, the content of which was 32.37 µg/mg, 10.59 µg/mg, 8.70 µg/mg, and 6.22 µg/mg respectively.&#x0D; Conclusions. Using the HPLC method determined the amino acids in the herb of Epilobium angustifolium L. The concentrations of L-aspartic acid, L-glutamic acid, L-arginine, L-alanine and L-phenylalanine are predominate among free and bound amino acids in the Epilobium angustifolium L. herb. The result shows that Epilobium angustifolium L. is the source of amino acids, so the use of this plant raw material for new remedies is possible in the future

Assessment of quality and safety of meats from various animal species in the Shuchinsk-Burabay resort zone, Kazakhstan

Aim:This study aimed to determine the food safety and protein adequacy of meats from various animals in the Shuchinsk-Burabay resort zone.Materials and Methods:Samples of meat were collected from markets “Zhomart” and “Kausar.” Two hundred and ninety-eight samples of meat were obtained: beef - 166, horse - 42, pork - 67, mutton - 8, and poultry - 15. From each carcass, part of the carcass, point samples were taken to form an average sample and conduct research. Analyses used inductively coupled plasma mass spectrometry and liquid chromatography. The determination of amino acid composition was carried out following the chromatographic method for determining the amino acid composition of meat proteins.Results:The quality of meats from the resort zone was lower than recommended due to the reduced content of essential amino acids, including valine, lysine, isoleucine, and phenylalanine. Concentrations of heavy metals and radionuclides did not exceed maximum permissible limits, and trace concentrations of arsenic, cadmium, and lead were detected in all meat samples, except beef. The latter meat showed increased concentrations of mercury.Conclusion:In terms of quality indicators, all types of meat met standards; however, pork protein displayed the most favorable amino acid composition, both for content of essential amino acids and the ratio of amounts of essential to non-essential amino acids. For content of heavy metals, poultry and pork meats were safest.

Variability Russian Rice Varieties in Essential Amino Acids Composition

Rice contains antioxidants, minerals, carbohydrates, lipids, vitamins that make it a functional food. A dozen times higher content of antioxidants was noted in black-grain and red-grain rice varieties as compared to white-grain ones. In terms of protein content, color and white-grain varieties do not differ significantly. The protein composition largely determines the nutritional value and functional properties, as well as the texture and taste of rice. Although rice contains less protein than many cereals, its biological value is highest due to balanced composition of amino acids. Nutritional value of different rice varieties varies greatly due to different amino acid composition. Determination of amino acid composition was carried out on devices Kapel 105 - M. In the work used 50 collection samples of rice. The variability was studied and the sources were identified among domestic rice varieties. To create a new generation varieties with more balanced amino acid composition, it is necessary both to isolate sources with a high content of individual amino acids, and varieties with high characteristics in terms of content complex of amino acids, especially essential ones.

Research of quality indicators of confectionery paste with sunflower flour

Confectionery products are in constant steady demand among various categories of consumers. This group of foodstuffs has a low nutritional and high energy value, contains a large amount of fats and carbohydrates with an insignificant content of essential micronutrients. In this regard, research on the modernization of recipes and technologies for confectionery products enriched with essential nutrients, especially with the use of secondary products of food processing, remains relevant. The aim of the research was to study quality indicators of sunflower flour and confectionery paste produced from it. Material and methods. The main objects of the study were flour obtained from sunflower oil cake after extracting oil by cold pressing, and confectionery paste based on it. Measurement of mass fractions of moisture, sugar, fat, protein and fiber, determination of amino acid composition and viscosity, organoleptic and safety indicators (the content of toxic elements, mycotoxins and pesticides, sanitary and microbiological indicators) have been carried out. A study of the viscosity of the confectionery paste and rheological parameters have been performed. The shelf life of the finished product was determined using the "accelerated aging" technique, which was confirmed by test data under standard conditions. Results. The study of the chemical composition of sunflower flour showed that the content of protein fluctuate from 39.0 to 45.0%, fiber - from 10.0 to 15.0%, fat - from 7.0 to 12.0%, mass the proportion of moisture wa from 2.0 to 6.0%. The total content of essential amino acids in sunflower flour (6.64 g/100 g) was 16% of the total protein content. Based on the analysis of variance, mathematical models were obtained that described the dependences of the acid and peroxide values of sunflower flour on the processing temperature and the duration of its exposure; it was found that the optimal conditions for heat treatment of flour were the processing temperature - 60 °C, the exposure time - 30 min. The optimal dosage of prescription components has been established based on the organoleptic and rheological characteristics of the confectionery paste. A commodity assessment of the developed confectionery paste was carried out in terms of organoleptic and physic-chemical quality indicators. The studies of safety indicators, including microbiological ones, made it possible to conclude that the paste fully meets the requirements of the Technical Regulations of the Customs Union TR CU 021/2011 "On food safety". To predict the shelf life of the paste in order to save time, the accelerated aging technique at elevated temperature was applied, which includes an analysis of the quality of the sample using the Van't Hoff rule and the Arrhenius equation. The study results were extrapolated to normal storage conditions. A study of the storage of confectionery paste under normal conditions at standard temperature regimes confirmed the results obtained using the accelerated aging technique. Studies of the chemical composition and nutritional value of the developed confectionery paste showed that it contained fats, proteins, which are a source of essential amino acids, and mineral substances. The shelf life of confectionery paste with sunflower flour at a temperature of 20±3 °C and a relative humidity of no more than 75% for no more than 10 months has been established. Conclusion. The complex research carried out has demonstrated that the use of sunflower flour in the production of confectionery paste makes it possible to obtain a product with high consumer characteristics and increased nutritional value.

Amino acid composition and antioxidant properties of the enzymatic hydrolysate of calabash nutmeg (Monodora myristica) and its membrane ultrafiltration peptide fractions.

The aim of this work was to determine amino acid composition and in vitro antioxidant activities of Monodora myristica protein hydrolysate and its membrane ultrafiltration peptide fractions. The Alcalase hydrolysate was fractionated using ultrafiltration membranes to produce peptide sizes of <1, 1-3, 3-5, and 5-10kDa. The results showed that sequential fractionation resulted in higher glycine and glutamic acid and glutamine contents. Analysis of in vitro antioxidant properties showed that fractionation of the M. myristica hydrolysate led to significant (p<.05) improvements in 2,2-diphenyl-1-picrylhydrazyl radical scavenging, metal chelation activity, ferric reducing antioxidant power (FRAP), and hydroxyl (OH) radical scavenging activity. Linoleic acid oxidation was significantly (p<.05) attenuated by the peptide fractions. We conclude that peptide antioxidant activities were significantly (p<.05) improved by membrane fractionation, especially the 3-5kDa fraction. PRACTICAL APPLICATIONS: The use of protein hydrolysate fractions with potential to prevent oxidation, which can reduce shelf life of foods and cause degenerate diseases due to cell damage is proposed for Monodora myristica. The demand for natural products and negative health issues associated with artificial food ingredients have led to increased consumer preference for natural sources of antioxidants. The protein hydrolysate and membrane fractions produced in this work showed high antioxidant ability that could qualify them to replace toxic synthetic antioxidants in foods. Peptide fractions had better metal chelation than the hydrolysates, which is important because chelation of metal ions can decrease the amount of free iron available to participate in the Fenton reaction and ultimately decrease the formation of toxic free radicals. Incorporation of the peptides into foods will enhance scavenging of toxic free radicals that may form during storage, thereby improving product freshness and shelf life in addition to preventing human degenerative diseases.

Determination of Amino Acid Composition of Ganirelix Acetate in an Injectable Formulation by Pre-column Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate.

Ganirelix is a synthetic decapeptide linked with nine different amino acids. To understand the peptide amino acid sequence or primary structure, the first step is to determine the amino acid composition of the peptide which can be a determining factor for the peptide immunogenicity. Edman degradation is not a suitable analytical technique to identify amino acid sequence present in Ganirelix due to the absence of uncharged N-terminal amino group. To address this challenge, a pre-column derivatization method was developed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. In the present work, the Ganirelix active pharmaceutical ingredient present in the injectable formulation was isolated by fraction collection and further purified by flash chromatography. The amino acid composition of Ganirelix is assayed by carrying out acid hydrolysis with 6molL-1 hydrochloric acid solution containing 1% phenol at 100°C for 24h and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent solution, followed by determination of individual amino acids by reverse-phase chromatography using a C18 column. High resolution was achieved for the nine amino acid mixture. The amino acid composition results of temperature-stressed Ganirelix generic product and reference listed drug are in good agreement with the theoretical molar ratio of label information.

Proteomic Analysis of Beef Tenderloin and Flank Assessed Using an Isobaric Tag for Relative and Absolute Quantitation (iTRAQ).

Simple SummaryAmino acid composition is among the important indexes of the nutritional composition of meat nutrients. In this study, we performed a proteomic analysis of tenderloin and flank steaks from Simmental cattle using isobaric tags for a relative and absolute quantification (iTRAQ) approach. Seventeen amino acids were detected in tenderloin and flank steaks, including seven essential amino acids and 10 non-essential amino acids. A comparison of the expression patterns in steaks revealed 128 differentially expressed proteins (DEPs). Furthermore, 27 DEPs (p < 0.05) were subjected to Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.Herein, we performed a proteomic analysis of tenderloin and flank steaks from Simmental cattle using the isobaric tags for a relative and absolute quantification (iTRAQ) approach. We identified 17 amino acids in both steaks, and Gly, Cys, Ile, Lys, and Pro differed most in abundance between the steak types (p < 0.05). A comparison of the expression patterns in steaks revealed 128 differentially expressed proteins (DEPs), of which 44 were up-regulated and 84 were down-regulated. Furthermore, 27 DEPs (p < 0.05) were subjected to gene ontology (GO) analysis, and many were found to be related to oxidation-reduction, metabolism, hydrogen ion transmembrane transport, transport, the tricarboxylic acid (TCA) cycle, mitochondrial electron transport, and the conversion of nicotinamide adenine dinucleotide (NADH) to ubiquinone. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also implicated these DEPs in various signalling pathways, including oxidative phosphorylation, cardiac muscle contraction, the TCA cycle, biosynthesis, and the metabolism. These findings provide a new insight into key proteins involved in the determination of amino acid composition in beef.

Determination of Amino Acids Composition in Different Tissues of Whiting, Merlangus merlangus euxinus (Nordmann, 1840) from the Black Sea, Turkey

LC-MS/MS was used to determine the amino acid composition in muscle, ovary and liver of whiting, Merlangus merlangus euxinus, caught off the coast of Sinop province in the Black Sea. A total of 19 amino acids (AA) were found in the different samples. The essential amino acids (EAA) in the different tissues of whiting were 55.9% in the meat, 54.8% in the ovary and 51.7% in the liver of the total amino acids. The AA contents except for Arg, Glu, Pro and Tau in meat and ovary of whiting were not significantly different (P &amp;gt; 0.05), but the AA contents except for Cys, Tyr, Asp, Orn and Tau of these two tissues were significantly (P &amp;lt; 0.05) higher than the AA contents of the liver. These results showed that whiting ovaries have approximately as much protein as the meat.

Quantitative amino acids profile of monofloral bee pollens by microwave hydrolysis and fluorimetric high performance liquid chromatography

Bee pollen is an attractive resource in the field of alternative remedies and thanks to the content of carbohydrates, crude fibers, proteins and lipids must be considered as a supplementary food of high potential rate. In characterization of bee pollen with the aim to define its value in human nutrition, the amino acids profile is one of the most important attributes. In the present study, the determination of amino acids composition of different monofloral bee pollen samples was obtained by an approach combining microwave acidic hydrolysis (60 min at 150 °C instead of 22 h at 120 °C in conventional oven) followed by derivatization using 9-fluorenylmethylchloroformate (FMOC-Cl) and separation of amino acids derivatives using a Phenomenex Kinetex core-shell 5 μm C18 (150 x 4.6 mm i.d.) column under a ternary gradient elution. Separation of 19 amino acids was achieved in about 40 min and fluorimetric detection (λexc = 265 nm λem = 315 nm) allowed selective and sensitive quantitation with LOQ values ranging within 0.14–3.00 μg/mL. Interestingly, the present approach allowed determination of some amino acids e.g., tryptophan and trans-4-hydroxyproline that are often lost by other methods of analysis. Significant differences in the composition of the considered samples were found confirming the impact of botanical origin of the product on its nutritional value. Principal Component Analysis was applied to treat the obtained data, highlighting the importance for discrimination, of detecting low abundance amino acids. The proposed method can be used as an advantageous alternative to the existing ones for characterization of bee pollen as an important source of dietary proteins.

Wheat Gluten Amino Acid Analysis by High-Performance Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection.

The present chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0M hydrochloric acid (HCl) solution at 110°C for 24h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). In contrast to conventional methods, the analysis requires neither pre- or post-column derivatization nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24h and for losses of Ser during evaporation. Gradient conditions including an extra eluent allow multiple sequential sample analyses without risk of Glu accumulation on the anion-exchange column which otherwise would result from high Gln levels in gluten proteins.

Isolation and characterization of kurstakin and surfactin isoforms produced by Enterobacter cloacae C3 strain.

In this work, the extraction, structural analysis, and identification as well as antimicrobial, anti-adhesive, and antibiofilm activities of lipopeptides produced by Enterobacter cloacae C3 strain were studied. A combination of chromatographic and spectroscopic techniques offers opportunities for a better characterization of the biosurfactant structure. Thin layer chromatography (TLC) and HPLC for amino acid composition determination are used. Efficient spectroscopic techniques have been utilized for investigations on the biochemical structure of biosurfactants, such as Fourier transform infrared (FT-IR) spectroscopy and mass spectrometry analysis. This is the first work describing the production of different isoforms belonging to kurstakin and surfactin families by Ecloacae strain. Three kurstakin homologues differing by the fatty acid chain length from C10 to C12 were detected. The spectrum of lipopeptides belonging to surfactin family contains various isoforms differing by the fatty acid chain length as well as the amino acids at positions four and seven. Lipopeptide C3 extract exhibited important antibacterial activity against Gram-positive and Gram-negative bacteria, antifungal activity, and interesting anti-adhesive and disruptive properties against biofilm formation by human pathogenic bacterial strains: Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, and Candida albicans.

Protein Extraction from Gels: A Brief Review.

Protein gel electrophoresis is an important procedure carried out in protein studies. Elution and recovery of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are often necessary for further downstream analyses. The process involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (e.g., enzymes) for subsequent analyses. Investigators have extracted proteins from gels by a variety of techniques. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity, and other purposes. Protein yields ranging from nanogram levels to 100μg have been obtained. Here, we review some of the methods that have been used to elute proteins from gels.

Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach.

Qualitative and quantitative determination of amino acid composition using amino acid analysis (AAA) is an important quality attribute and considered an identity of therapeutic peptide drugs by the regulatory agencies. Although huge literature is available on pre- and post- column derivatization AAA methods, arriving at an appropriate hydrolysis protocol coupled with adequate separation of the derivatized/underivatized amino acids is always challenging. Towards achieving a facile and comprehensive protocol for AAA, the present work is geared towards developing a deeper understanding of the extent of hydrolysis of peptide, and the nature and stability of amino acids present in the peptide backbone. This defines the suitability of the method in meeting the end goals and the regulatory requirement. Analysis of historical data generated during the method optimization of AAA for icatibant acetate (ICT) using head space oven hydrolysis (HSOH) and microwave-assisted hydrolysis (MAH) methods helped in arriving at fast (< 1h) and efficient hydrolysis (0.9-1.1 of theoretical residue) conditions. Better separations for the natural and unnatural amino acids were achieved using 3.45 ≤ pH ≤ 10.85, and a column oven gradient program. This approach was useful in meeting the method quality attributes [resolution (Rs) > 2.0; plate count (N) > 5600; and USP tailing factor < 1.2] with a target analytical method profile of relative amino acid mole ratios (RAAMR) in the range of 0.9-1.1 for Ser, Oic, Tic, Hyp, Ala (Thi), Gly and Pro, and between 2.7 and 3.3 for Arg. The developed method was validated as per the ICH guidelines and is precise, accurate, linear and robust.

Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety of a novel fibrin(ogen)olytic serine protease from leaves of Leucas indica

The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme’s novel characteristic. Lunathrombase is an αβ-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125–0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.

ВИЗНАЧЕННЯ АМІНОКИСЛОТНОГО СКЛАДУ ТА МІКРОБІОЛОГІЧНИХ ПОКАЗНИКІВ ВАРЕНИХ КОВБАС

Аналіз структури харчування різних груп населення свідчить, що в даний час потреби в білках, в першу чергу тваринного походження, задовольняються лише на 80%.&#x0D; Для досліджень розробили рецептури варених ковбас із заміною частини м’ясної сировини гідратованою білоквмісною харчовою композицією (до 30%) та м’ясом птиці механічного обвалювання (до 30%).&#x0D; Для доведення доцільності використання розробленої композиції у складі варених ковбасних виробів провели визначення амінокислотного складу та вивчили її вплив на термін зберігання готових ковбас шляхом дослідження мікробіологічних показників. Доведено, що спрямоване застосування харчових композицій у технології варених ковбас дозволяє нормалізувати загальний хімічний і амінокислотний склад, забезпечуючи мікробіологічну стабільність готової продукції.&#x0D; Встановлено, що заміна м’ясної сировини ФХК в кількості 30% (характерна для дослідного зразка №1) дозволяє зберегти амінокислотний склад продукту, а додаткове внесення МПМО (характерне для дослідного зразка №2) призводять до його зменшення, особливо таких незамінних амінокислот як ізолейцин та валін.&#x0D; Розроблені композиції сприяють зниженню залишкової мікрофлори та пригнічують зростання мезофільно-аеробних і факультативно анаеробних мікроорганізмів в готових виробах, що пов’язано з їх нижчим початковим мікробіологічним обсіменінням порівняно із м’ясною сировиною.&#x0D;