Determination Of Protein Content Research Articles

Potensi Ekstrak Enzim Daun Pepaya (Carica papaya L.) Sebagai Agen Fibrinolitik Dengan Metode Clot Lysis in Vitro

Stroke is a disease caused due to blockage of blood vessels. Health department in 2013 stated that 15,4% of people died from strokes. One of the treatment therapies that can be used is fibrinolytic. Fibrinolytic agents have a role in lysing blood clots, these agents can be obtained from microbes, plants and animals. This study aims to determine the protein content contained in papaya leaf enzyme extract (Carica papaya L.), and to determine the potential of the enzyme extract at certain concentrations as an alternative to natural fibrinolytic drugs. The study began with plant determination, sampling, enzyme extraction using a blender, purification of the extract by precipitation using ammonium sulphate, protein content determination using the Lowry method, and testing the fibrinolytic activity of the clot lysis method in vitro. Three series of test concentrations were made, namely 20, 40, and 80%. The results of the research on papaya leaf extract showed that the protein content in purification pellet 1 was 196,22 µg/ml and purification pellet 2 was 306,33 µg/ml. In papaya leaves, fibrinolytic activity was proven with the optimal percentage of blood clot lysis in purification pellet 2 with a concentration of 80% which was 79%.

Comprehensive Phytochemical Analysis and Bioactivity Evaluation of Padina boergesenii: Unveiling Its Prospects as a Promising Cosmetic Component.

Marine macroalgae, such as Padina boergesenii, are gaining recognition in the cosmetics industry as valuable sources of natural bioactive compounds. This study aimed to investigate the biochemical profile of P. boergesenii and evaluate its potential as a cosmetic ingredient. Fourier-transform infrared (FTIR), gas chromatography-mass spectrometry (GCMS), and high-resolution liquid chromatography-mass spectrometry quadrupole time-of-flight (HRLCMS QTOF) analyses were employed to assess the functional groups, phycocompounds, and beneficial compounds present in P. boergesenii. Pigment estimation, total phenol and protein content determination, DPPH antioxidant analysis, and tyrosinase inhibition assay were conducted to evaluate the extracts' ability to counteract oxidative stress and address hyperpigmentation concerns. Elemental composition and amino acid quantification were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES) and HRLCMS, respectively. FTIR spectroscopy confirmed diverse functional groups, including halo compounds, alcohols, esters, amines, and acids. GCMS analysis identified moisturizing, conditioning, and anti-aging compounds such as long-chain fatty alcohols, fatty esters, fatty acids, and hydrocarbon derivatives. HRLCMS QTOF analysis revealed phenolic compounds, fatty acid derivatives, peptides, terpenoids, and amino acids with antioxidant, anti-inflammatory, and skin-nourishing properties. Elemental analysis indicated varying concentrations of elements, with silicon (Si) being the most abundant and copper (Cu) being the least abundant. The total phenol content was 86.50 µg/mL, suggesting the presence of antioxidants. The total protein content was 113.72 µg/mL, indicating nourishing and rejuvenating effects. The ethanolic extract exhibited an IC50 value of 36.75 μg/mL in the DPPH assay, indicating significant antioxidant activity. The methanolic extract showed an IC50 value of 42.784 μg/mL. Furthermore, P. boergesenii extracts demonstrated 62.14% inhibition of tyrosinase activity. This comprehensive analysis underscores the potential of P. boergesenii as an effective cosmetic ingredient for enhancing skin health. Given the increasing use of seaweed-based bioactive components in cosmetics, further exploration of P. boergesenii's potential in the cosmetics industry is warranted to leverage its valuable properties.

Optimum pH Buffer of Phosphate and Carbonate on The Crude Extraction of Uricase Enzyme from Goat Liver

Uricase enzyme (urate oxidase) is an enzyme that catalyze the oxidation of uric acid in the presence of oxygen to produce allantoin, carbon dioxide (CO2) and hydrogen peroxide (H2O2). Human and primates do not have this enzyme while other mammals have it in the liver therefore the uricase enzymes are extracted from goat liver use a buffer that is compatible with the human buffer system. The type of buffer selected adjusted at the appropriate pH. The optimum uricase pH ranged from 7.5 to 9.5. The selection of buffer type is adjusted to the human buffer system. The purpose of the study was to determine the effect of the type and pH of the extraction buffer on the total activity, protein total and specific activity of crude uricase. The types of buffer selected are phosphate buffer and carbonate buffer, while the selected pH is 7.5; 8.5; and 9.5. The method used is enzyme extraction, then determination of enzyme activity and protein content to determine the specific activity of the enzyme. The results obtained the highest total enzyme activity at pH 8.5 both in carbonate buffer (0.0481 U/mL) and phosphate buffer (0.0383 U/mL). The highest protein total in carbonate buffer was at pH 9.5 (4.55 mg/mL) while the highest value was in phosphate buffer pH 8.5 (4.1 mg/mL). The specific activity of uricase pH 8.5 was carbonate buffer (0.0114 U/mg) and phosphate buffer (0.0094 U/mg). The highest uricase specific activity value was at pH 8.5 for both carbonate and phosphate buffer types and in the long term it is used as a gout therapy.

Structural characterizations and α-glucosidase inhibitory activities of four Lepidium meyenii polysaccharides with different molecular weights

Four polysaccharides (MCPa, MCPb, MCPc, MCPd) were obtained from Lepidium meyenii Walp. Their structures were characterized by chemical and instrumental methods including total sugar, uronic acid and protein content determination, UV, IR and NMR spectroscopy, as well as monosaccharide composition determination and methylation analyses. Four polysaccharides were a group of glucans with different molecular weights ranging from 3.12 to 14.4 kDa, and shared a similar backbone chain consisting of (1→4)-glucose linkages with branches attached to C-3 and C-6. Furthermore, bioactivity assay showed that MCPs had concentration-dependent inhibitory activity on α-glucosidase. MCPb (Mw = 10.1 kDa) and MCPc (Mw = 5.62 kDa) with moderate molecular weights exhibited higher inhibitory activity compared with MCPa and MCPd.Graphical

Thermal characterization of leathers tanned by metal salts and vegetable tannins

In this work, the thermal properties of vegetable (mimosa), metal salt (chromium) and mixed (chromium-mimosa and aluminum-myrobalan) tanned bovine leathers were studied to understand the thermal decomposition processes with the aim of possible utilization of various leather wastes by thermal conversion. Thermogravimetry/mass spectrometry (TG/MS) and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) measurements were carried out to compare the thermal behavior and reveal interactions between the leather and various tannins. Bovine parchment was used as an untanned reference material. The tanning agent ratios were estimated by determination of ash, nitrogen and protein contents of the samples. It was found that ash yield is a good indicator for the inorganic content, while the protein content is inversely proportional to the total tannin content in leathers. The thermal decomposition of the samples as a function of the temperature was performed using TG/MS. The differences between the samples are clearly recognizable on the thermogravimetric curves and the mass spectrometric evolution profiles of the products. Among the tanning agents, chromium had the largest impact on the thermal decomposition process. Therefore, additional TG/MS experiments were carried out on chromium-treated parchment samples and the catalytic effect of chromium was demonstrated on the evolution curves of several products. It was established that hydrolysis of the amide bonds of the polypeptide chain was retarded resulting in less ammonia formation, and higher yield of water. On the other hand, higher amounts of hydrogen cyanide and other nitriles were released in the presence of chromium sulfate. The decomposing chromium sulfate released oxygen beside sulfur dioxide, which dehydrogenated certain amino acid residues leading to the enhanced formation of nitriles and water. The volatile compounds of the decomposition were determined by Py-GC/MS at 400 and 600 °C. The diketopiperazines (DKPs) dominated the pyrograms at both temperatures, but the ratio of gaseous and other products of smaller molecular mass was increased at higher pyrolysis temperature and in the presence of metal tanning agents. The chromium-treated parchment samples were also studied by Py-GC/MS. The relationship between the chromium content and the formation of certain decomposition products has been confirmed. Principal component analysis (PCA) was performed using the Py-GC/MS data of leathers and parchment. The PCA results show clear separation between the chromium-tanned, vegetable-tanned and mix-tanned leathers as well as parchment.

Variable selection methods to determine protein content in paddy using near-infrared hyperspectral imaging

Variable selection methods to determine protein content in paddy using near-infrared hyperspectral imaging

Диагностика, лечение и профилактика синдрома перенапряжения у спасателей МЧС России

Summary. The aim of the study is to evaluate modern scientific views on the problem of diagnosis, treatment and prevention of syndromes associated with physical and emotional overstrain in rescue workers of the Russian Ministry of Emergency Situations. Study materials and methods. An analysis of domestic and foreign literature data was performed. The search was performed using the Google Academy search engine and electronic databases PubMed, MEDLINE, Embase, Scopus, Web of Science, eLIBRARY for the period from 2017 to 2022. Results of the study and their analysis. Currently, early diagnosis of overexertion syndrome (overtraining) is quite complicated. It is caused by the absence of clear diagnostic criteria, gradual development of the pathology and nonspecificity of its symptoms. That is why a complex approach is important for timely and high-quality diagnosis of overexertion, which includes a set of diagnostic measures, such as collecting anamnesis and visual examination, completing psychological questionnaires, examination of hormone levels at rest and after exercise tests, examination of blood biochemical characteristics (determination of total protein concentration, lipoproteins, glucose, cholesterol, etc.) and instrumental examination. The main ways to fight against overtraining syndrome are emotional rest, gentle training regime, adequate replenishment of energy and plastic substrates, and a sufficient amount of sleep. Usage of manual therapy, therapeutic physical training and physiotherapy is also possible.

Determination of corn protein content using near-infrared spectroscopy combined with A-CARS-PLS.

In order to quickly and accurately determine the protein content of corn, a new characteristic wavelength selection algorithm called anchor competitive adaptive reweighted sampling (A-CARS) was proposed in this paper. This method first lets Monte Carlo synergy interval PLS (MC-siPLS) to select the sub-intervals where the characteristic variables exist and then uses CARS to screen the variables further. A-CARS-PLS was compared with 6 methods, including 3 feature variable selection methods (GA-PLS, random frog PLS, and CARS-PLS) and 2 interval partial least squares methods (siPLS and MWPLS). The results showed that A-CARS-PLS was significantly better than other methods with the results: RMSECV=0.0336, R2 c=0.9951 in the calibration set; RMSEP=0.0688, R2 p=0.9820 in the prediction set. Furthermore, A-CARS reduced the original 700-dimensional variable to 23 variables. The results showed that A-CARS-PLS was better than some wavelength selection methods, and it has great application potential in the non-destructive detection of protein content in corn.

Bacteriological and Physicochemical Study of Retailed Cow Milk collected from Different Locations in Birnin Kebbi

Abstract
 Milk is an excellent source of both major and minor components needed to meet the nutritional requirements of the human body. The current study was conducted to evaluate the bacteriological and physicochemical quality of cow milk collected from various locations in Birnin Kebbi. A total of twenty-four (24) milk samples (six from each location) were purchased. All samples were subjected to bacteriological analysis using total plate count, and physicochemical composition analysis, which included PH, moisture content, fat content, protein content, and mineral determination (calcium, sodium, and potassium). The mean range for Total Plate Count is 1.83x106-3.42x106 CFU/ML. Furthermore, bacteria isolated and identified from milk samples include Streptococcus spp, Shigella spp, Escherichia coli, Bacillus spp, Staphylococcus aureus, and Proteus spp. The physicochemical analysis results indicated that the mean range is PH (4.87±0.04-5.52±0.05) Moisture content (82.22±0.60-87.50±2.16), Fat content (0.56±0.02-1.84±0.01), Protein content (11.27±4.4-12.55±4.99), Calcium (173.83±4.43-475.67±22.24), Sodium (11.83±4.26- 111±12.44) and Potassium (131±9.12-596±6.16) respectively. The study's findings highlight the poor bacteriological quality of milk, as evidenced by a higher Total Plate Count. Moreover, the presence of pathogenic microbes in milk, such as Bacillus spp, Shigella spp, and Escherichia coli, is likely to pose a serious public health risk. As a result, it is recommended that proper hygiene practices be implemented, as well as the incorporation of effective monitoring throughout the production to delivery chain.

Combination of near-infrared spectroscopy and key wavelength-based screening algorithm for rapid determination of rice protein content

Rice taste quality is one of the most important factors influencing rice marketing and distribution, and rice with a high taste quality is more popular with consumers and has a higher price. Accurate and rapid determination of rice protein content helps to assess the rice taste quality and aids in marketing. In this study, NIR spectra of 84 rice samples combined with partial least squares regression (PLSR) were used to model protein content, and different selection algorithms based on key wavelengths (competitive adaptive reweighted sampling, CARS; Monte-Carlo uninformative wavelength elimination, MC-UVE; random frog, RF) were used to understand the accuracy of NIR spectra in predicting the rice protein content. Our results showed that the R2P and RPD of the original full-spectrum PLSR model were 0.83 and 1.95, respectively. After the second-order derivative preprocessing, the R2P and RPD of the full spectrum were improved to 0.95 and 4.14. Both CARS and MC-UVE increased the R2P of the PLSR model to 0.97 and the RPD to 5.57 and 5.65, respectively. R2C and R2CV in the PLSR model based on CARS algorithm were 0.93 and 0.91, respectively. The CARS algorithm had excellent results in predicting the rice protein content.

Anchovy’s protein as a potential precursor of Angiotensin-I Converting Enzyme (ACE) inhibitory peptide and Dipeptidyl Peptidase-IV (DPP-IV) inhibitory peptide by an in silico approach

Protein from fish is known as the precursor of biologically active peptides that exert various health benefits such as antihypertensive, antitumor, and immunomodulatory properties. Hence, this study aimed to perform an extraction of anchovy, LC-MS/MS analysis and in silico evaluation of the major proteins in anchovies as potential precursors of biologically active peptides in addition to determining whether such peptides can be released by selected proteolytic enzymes. Anchovy was subjected to protein extraction followed by total soluble protein concentration determination using Bradford assay. The sample was subjected to in-solution trypsin digestion, which was then analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A bioinformatic approach by PEAKS Studio was used to identify the protein. A total of four anchovy’s proteins which are myosin light chain 1, V(D)J recombination-activating protein, 60 kDa chaperonin and heat shock protein 90AA1 were identified. Then, the identified proteins were subjected to in silico approach using the BIOPEP database. The biological potential of the theoretically released angiotensin-I converting enzyme (ACE) inhibitory peptides and dipeptidyl peptidase (DPP)-IV inhibitory peptides were predicted by determining the frequency of occurrence of fragments with a given activity. 60 kDa chaperonin and heat shock protein 90AA1 predicted the highest number of biological activities for ACE inhibitory peptides (284 and 264 fragments) and DPP-IV inhibitory peptides (395 and 409 fragments). The most promising enzyme for the generation of bioactive peptides from anchovy protein was anticipated to be pepsin (pH > 2), which theoretically released a high number of DPP-IV inhibitory peptides and ACE inhibitory peptides through the action of in silico proteolysis. Overall, this work highlighted that anchovy protein could be a promising precursor of bioactive peptides that have ACE and DPP-IV inhibitory activities for developing functional food or nutraceutical products.

Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 μg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC–MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Grain yield and protein content in different ripening varieties of soft winter wheat using various types and doses of fertilizers

Purpose. To study of yield formation and protein content in the grain of different ripening varieties of soft winter wheat under the condition of using various types and doses of fertilizers. Methods. Field and laboratory (determination of protein content), calculation (collection of protein), mathematical and statistical. Results. It was found that different fertilization systems reliably increased the grain yield of soft winter wheat. The use of N75 increased this indicator by 1.2 times and N150 by 1.4 times in the varieties studied, compared with the no-fertilizer variant. Yield with incomplete return of phosphorus-potassium fertilizer was only 2–3% lower than with complete mineral fertilizer. With the nitrogen-phosphorus and nitrogen-potassium fertilization systems, the yield was 5–7% higher than with the nitrogen system. At the same time, this indicator was 6% lower than in the full mineral fertilizer version of the trial. The use of 75 kg/ha nitrogen fertilizer per year increased the protein content to 13.5%, or by 10% compared to the control. In the double dose nitrogen fertilizer variant, the protein content increased to 14.2% or by 15%. The use of nitrogen fertilizers with phosphorus-potassium only contributed 2–4% to the increase of this indicator. Cultivation of winter wheat under the condition of application of N75 increased the collection of protein up to 724 kg/ha or by 33%, and with a double dose of nitrogen fertilizer – up to 848 kg/ha or by 55% compared to the option without fertilizer. In the N75P30K40 variant, protein yield increased by 10% compared to the nitrogen system. The application of a complete mineral fertilizer (N150P60K40) increased this indicator by 12%. Conclusions. It was found that the use of N75P30K40 increased the grain yield of soft winter wheat by up to 5.9 t/ha, or 7%, compared to the option where only 75 kg/ha of nitrogen fertilizer was applied. Yield development in soft winter wheat varies considerably from variety to variety. For example, this indicator was 35% higher in the ‘KWS Emil’ variety than in the ‘Prino’ line. In addition, the grain yield of both varieties is reliably influenced by the weather conditions during the growing season. The protein content of soft winter wheat varies considerably depending on the variety and the weather conditions. When growing soft winter wheat varieties, the protein content can vary by 12.8–15.1%. Weather conditions during the growing season can change this indicator by 13.1–14.7%. The protein content index in the grain of the ‘Prino’ line is significantly higher than that of the ‘KWS Emil’ variety. According to the protein yield per hectare indicator, the ‘KWS Emil’ variety (896 kg/ha) has a significant advantage over the ‘Prino’ line (774 kg/ha).

Near-infrared hyperspectral imaging for determination of protein content in barley samples using convolutional neural network

Near-infrared hyperspectral imaging for determination of protein content in barley samples using convolutional neural network

Validasi Metode Analisis Penetapan Kadar Protein Ekstrak Ikan Gabus dengan Metode Lowry dan Bromocresol Green

Snakehead fish is one of the biological resources with high economic value because it contains high albumin levels. Analytical methods for the determination of protein content in snakehead fish are available not been validated. This study aims to obtain a comparison of the accuracy, repeatability, intermediate precision, and linearity between the Lowry and Bromocresol-green methods in determining albumin levels from snakehead fish extracts. This study was performed with the SHIMADZU UV-1280 spectrophotometer at a wavelength of 778 nm for the lowry method and 638 nm for the bromocresol-green method. The results of the study show that all parameters meet the requirements for accepting validation. The results were tested statistically with the independent t-test, the p-value was 0.172 (p> 0.05) the accuracy between the Lowry and Bromocresol-green methods did not have a significant difference, that the Lowry and Bromocresol green methods had the same ability to determine accuracy parameters and precision parameters, the p-value is 0.000 (p

A non-destructive determination of protein content in potato flour noodles using near-infrared hyperspectral imaging technology

Hyperspectral imaging (HSI) with the near-infrared (NIR) bands (900–2250 nm) was employed to investigate the non-destructive prediction of protein content in potato flour noodles. The protein content (8.906–10.515 %) of 120 potato flour noodles was studied to establish the prediction model. Partial least squares regression (PLSR) model was established based on the spectra of potato flour noodles to predict the protein content showing high performance. After optimization, orthogonal signal correction (OSC) was used to preprocess the original spectra, and the competitive adaptive reweighted sampling algorithm (CARS) was chosen to select characteristic wavelengths, therefore, OSC-CARS-PLSR was established. Next, 77 samples were selected as the calibration set, and the remaining 38 samples were used as the prediction set. The coefficient of determination (R2) and the root mean square error (RMSE) were used to evaluate the performance of the model. OSC-CARS-PLSR showed a high performance with R2 values of 0.9606 and 0.8925 and RMSE values of 0.070 % and 0.1385 % in the calibration set and prediction set, respectively. The visualization image was used to identify protein distribution in potato flour noodles. Overall, the results indicate that HSI technology could accurately predict the protein content in potato flour noodles providing a rapid and non-destructive method to detect protein and other compositions in grains and foods.

Nondestructive determination and visualization of protein and carbohydrate concentration of Chlorella pyrenoidosa in situ using hyperspectral imaging technique

As a new food resource approved by the National Health Commission of China, Chlorella pyrenoidosa (C. pyrenoidosa) has become commercialized in recent years with the advantages of comprehensive nutrition, especially rich in protein content. Monitoring the growth information of C. pyrenoidosa is crucial for optimizing the culture environment and increasing microalgae yield. Traditional methods for the detection of microalgae bioproducts are time-consuming and expensive. In this study, a fast visual and non-invasive method based on visible/near infrared (VIS/NIR) hyperspectral imaging (HSI) combined with chemometric methods was developed to predict the biomass, carbohydrate and protein in the cultures of C. pyrenoidosa. Twelve data preprocessing approaches, 3 feature selection methods, and 4 calibration models were used to establish and optimize the estimation models. The prediction results showed that the effects of autoscaling preprocess combined with CARS-MLR for biomass (R2p = 0.9788, RPD = 7.6503), wavelet transform (WT) combined with iRF-RFR for carbohydrate (R2p = 0.9935, RPD = 27.0385), and S-G preprocess combined with SA-RFR (R2p = 0.9677, RPD = 12.9928) for protein obtained the best performances, respectively. Moreover, visualization maps of the distribution and abundance of these components in the liquid suspension of C. pyrenoidosa were obtained based on the optimal models. This study showed that HSI technology combined with chemometric methods can accurately predict the biomass, carbohydrate, and protein contents of C. pyrenoidosa in situ, which has the potential as a fast and nondestructive approach for monitoring microalgal growth information.

Existence of a superior polysorbate fraction in respect to protein stabilization and particle formation?

Biologicals including monoclonal antibodies are the current flagships in pharmaceutical industry. However, they are exposed to a multitude of destabilization conditions like for instance hydrophobic interfaces, leading to reduced biological activity. Polysorbates are commonly applied to effectively stabilize these active pharmaceutical ingredients against colloidal stress. Nevertheless, chemical instability of polysorbate via hydrolysis or oxidation results in degradation products that might form particles via phase separation. Polysorbates are mixtures of hundreds of individual components, and recently purer quality grades with reduced variations in the fatty acid composition are available. As the protective function of polysorbate itself is not completely understood, even less is known about its individual components, raising the question of the existence of a superior polysorbate species in respect to protein stabilization or degradation susceptibility. Here, we evaluated the protective function of four main fractions of polysorbate 20 (PS20) in agitation studies with monoclonal antibodies, followed by particle analysis as well as protein and polysorbate content determination. The commercially-available inherent mixtures PS20 high purity and PS20 all-laurate, as well as the fraction isosorbide-POE-monolaurate showed superior protection against mechanical–induced stress (visual inspection and turbidity) at the air–water interface in comparison to sole sorbitan-POE-monolaurate, -dilaurate, and -trilaurate. Fractions composed mainly of higher–order esters like sorbitan-POE-dilaurate and sorbitan-POE-trilaurate indicated high turbidities as indication for subvisible and small particles accompanied by a reduced protein monomer content after agitation. For the isosorbide-POE-monolaurates as well as for the inherent polysorbate mixtures no obvious differences in protein content and protein aggregation (SEC) were observed, reflecting the observations from visual appearance. However, absolute polysorbate concentrations vary drastically between different species in the actual formulations. As there are still open questions in respect to protein specificity or regarding mixtures versus individual components of PS20, further studies must be performed, to gain a better understanding of a “generalized” stabilizing effect of polysorbates on monoclonal antibodies. The knowledge of the characteristics of individual polysorbate species can have the potential to pave the way to superior detergents in respect to protein stabilization and/or degradation susceptibility.

CHANGE OF SPONTANEOUS OXIDATIVE MODIFICATIONS OF SPERMОPLASM'S PROTEINS IN PATIENTS WITH VARICOCELE AND ASTHENOZOOSPERMIA

Oxidative stress plays one of the leading roles in the pathogenesis of varicocele, which affects the quantity and quality of seminal fluid. The research of ejaculate are currently based mainly on chemical-microscopic methods. Biochemical parameters of sperm are determined in insufficient volume, therefore, the introduction of new biochemical markers in order to diagnose a decrease in male fertility is an urgent issue. Oxidatively modified proteins can be considered as a marker of decreased fertility in men with varicocele. The aim of the work was to research the carbonylation of spermоplasm proteins in patients with varicocele II and III degrees with accompanying asthenozoospermia in the anamnesis. Material and methods. The study shows results of 60 men medical examination. The patients weredivided into two groups. The first group consisted of 30 men diagnosed with grade II and III varicocele with accompanying asthenozoospermia. The second group consisted of 30 patients with normozoospermia and without impaired reproductive function. The material for the study was spermoplasm. Spermoplasm was obtained by centrifugation of sperm samples for 10 minutes at a speed of 1000 rpm. Chemical microscopic examination of the ejaculate was carried out on a sperm analyzer (AFS-500-2, Russia) and light microscopy (microscope MS 100 X, Austria). Determination of protein concentration in spermoplasm was carried out by biuretic method with commercial kits of the company (Mindrey, China) on the biochemical analyzer Mindrey BS 120 (China). Determination of oxidative modification of proteins in spermoplasm was carried out by the method of R.L. Levine in modification by E.E. Dubinina. Results. There was a 2-fold increase in ADNPhG at λ 356, and λ 430 by 3.2 times in patients of the first group. It was also noted that the increase in КDNPhG at λ 370 increased by 2 times, and λ 530 by 3.9 times in the spermoplasm, in contrast to patients of the control group. It was also noted that the increase in KDNPhG at λ 370 increased by 2 times, and λ 530 by 3.9 times in the spermoplasm, in contrast to patients in the control group. Conclusion. An increase in oxidatively modified proteins in patients with varicocele may be associated with an increase in oxidative processes, a decrease in the antioxidant defense of the body and damage to the DNA of spermatozoa, which leads to a violation of its functions.

Towards absolute quantification of protein genetic variants in Pisum sativum extracts

In recent years, several studies have used proteomics approaches to characterize genetic variant profiles of agricultural raw materials. In such studies, the challenge is the quantification of the individual protein variants. In this study a novel UPLC-PDA-MS method with absolute and label-free UV-based peptide quantification was applied to quantify the genetic variants of legumin, vicilin and albumins in pea extracts. The aim was to investigate the applicability of this method and to identify challenges in determining protein concentration from the measured peptide concentrations. Analysis of the protein mass balance showed significant losses of proteins in extraction (37%) and of peptides in further sample preparation (69%). The challenge in calculating the extractable individual protein concentrations was how to deal with these insoluble peptides. The quantification approach using average amino acid concentrations in each position of the sequence showed most reproducible results and allowed comparison of the genetic protein composition of 8 different cultivars. The extractable protein composition (μM/μM) was remarkably similar for all cultivar extracts and consisted of legumins A1 (12.8 ± 1.2%), A2 (1.1 ± 0.4%), B (9.9 ± 1.6%), J (7.5 ± 1.0%) and K (10.3 ± 2.1%), vicilin (15.2 ± 1.7%), provicilin (15.7 ± 2.5%), convicilin (9.8 ± 0.8%), albumin A1 (7.4 ± 2.0%), albumin 2 (10.0 ± 1.5%) and protease inhibitor (0.4 ± 0.4%).