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A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding
Abstract
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
... Subsequently, the protein extract was treated with a 1 % proteinase inhibitor (Sigma-Aldrich, Cat. No.: P9599, St. Louis, Missuori, USA) and stored at − 20 • C. Protein concentrations were determined using bovine serum albumin as a standard (Bradford, 1976). ...
... Subsequently, 150 μL of the protein extract was incubated in a mixture consisting of 650 μL reaction buffer (20 mM TRIS-HCl pH 8.0, 0.5 mM EDTA), 100 μL of 0.2 mM NADH, and 100 μL of 0.4 mM GSNO. The protein concentration was determined using the Bradford protein assay (Bradford, 1976). The GSNOR activity data were expressed as nmol mg − 1 protein. ...
... The protein content in the filtrate was determined according to the Bradford method, using bovine serum albumin (BSA) as a standard. 35 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) was run to confirm the previous results of the activity assay. ...
Enzyme immobilization has been extensively studied to access higher enzyme stability and recyclability, enable continuous operations, and thus increase overall productivity in lab- and technical-scale processes. Among different immobilization methods,...
... After another precipitation with MgCl 2 and ultracentrifugation (300,000 ✕ g for 1.5 h at 4°C), the resulting pellet containing the BBMVs was resuspended in 50 µL of MET-PI buffer. Total protein was quantified using Bradford method using bovine albumin serum as standard [23]. We used two different approaches to validate our method of BBMV isolation: (i) enrichment of aminopeptidases through enzymatic assay; and (ii) separation of BBMV proteins by SDS-PAGE and visualization of band enrichments in silver-stained gels. ...
We investigated the protein composition of the brush border membrane of larval Frankliniella occidentalis (western flower thrips), an agriculturally significant crop pest and vector of plant pathogens. We developed a protocol for purifying brush border membrane vesicles (BBMVs) from first-instar larvae (L1) bodies and identified their protein composition by LC-MS/MS. From 2544 proteins identified, 469 were predicted to be secreted and part of the inner and outer plasma membrane leaflet using cell-localization prediction tools and homology with reviewed proteins in the UniProt database. Comparison to thrips tissue-specific proteomes revealed that 371 and 263 of the identified BBMV plasma membrane and secreted proteins matched the larval gut and adult salivary glands, respectively. Annotations of most of the proteins inferred ‘catalytic activity’ (56.3%) and ‘binding’ (49.6%), with an overrepresentation of proteins involved in protein digestion, specifically serine proteases, lipid transport, and ATPase activity. Bioinformatic-enabled comparisons to thrips tissue-specific proteomes and transcriptomes enabled us to predict the secretome and the plasma membrane proteins of larval thrips’ gut epithelial cells, providing new targets for thrips control.
... The proteins were quantified using standard Bradford's assay procedure (Bradford 1976). The protein digestion protocol was as per Promega: Technical Bulletin TB373 (https:// www. ...
Proteomics have proven advantage in drug and disease physiology characterization. Here the polyherbal formulation was administered daily via oral gavage in two groups of Six Sprague Dawley diabetic rats at the doses of 250 mg/kg and 500 mg/kg body weight for 21 days to understand its antidiabetic potential with proteomics approach. Blood sugar levels were monitored weekly during experimentation. The concurrent control group receiving 10 mL/kg water was also maintained. Rats were examined regularly for signs of toxicity and mortality and underwent detailed clinical examinations prior to initiation and weekly thereafter. Body weight and food consumption were recorded weekly. The anti-hyperglycaemic effect of the formulation was estimated from blood glucose levels weekly. There was no observed mortality or adverse clinical signs among the rats exposed to the standard drug and formulation. Streptozotocin caused a significant weight loss in rats, while treatment with formulation at 250 and 500 mg/kg b.w. concentrations and Glibenclamide as a standard drug; restrained the decrease in body weight. The streptozotocin-induced diabetic rats exhibited a sharp elevation in blood glucose levels. The blood glucose levels were significantly lowered in a dose dependent manner post formulation treatment, in comparison to the control group. Treatment with formulation, standard, and streptozotocin did not induce any remarkable gross pathological alterations in any of the organs/tissues of rats. In proteomics analysis, in formulation treatment groups ECM and Circadian entrainment pathways were activated which are in line with the objective of normalization of altered metabolism in diabetes.
... Samples were then frozen at -20ºC to disrupt haemocyte membranes (Wilson et al., 2001). The Bradford method was used to quantify the protein concentration of the haemolymph samples (Bradford, 1976). ...
Tannins are among the most abundant secondary metabolites synthesized by plants. Agelastica alni L., 1758 (Coleoptera: Chrysomelidae) is a critical forest pest. This study investigated the effect of Paenibacillus alvei (Cheshire & Cheyne) Ash et al. (Bacillii: Paenibacillaceae) and tannins against A. alni larvae. The larvae were collected from the Çayeli district of Rize province in 2022. In the feeding experiments, artificial diets containing 1.25%, 2.5% and 5% tannins were prepared. 100 and 200 µl of P. alvei were applied to the infected groups. Nutritional indices, pupal masses, phenoloxidase activities, antioxidant enzyme activities and mortality rates of larvae fed with different diets were studied. Relative consumption rate (RCR) increased with tannin concentration in all groups. Relative growth rate (RGR) increased with rising tannin concentrations across all groups. In the infected groups, the increase in tannin concentration caused a decrease in developmental time. While superoxide dismutase and phenoloxidase activities of uninfected larvae decreased with tannin concentration, catalase and glutathione peroxidase activities of larvae increased. In infected larvae, catalase activity decreased with increasing tannin concentration. The dose of P. alvei caused an increase in superoxide dismutase and phenoloxidase activities, but did not affect catalase and glutathione peroxidase activities. The diet containing 5% tannic acid had the lowest mortality rate.
... Protein concentration was measured by the Bradford method [17], utilizing bovine serum albumin (BSA) as the standard. SDS-PAGE was performed based on Laemmli's well-established protocol [18]. ...
Laceyella sacchari strain BK-TM, isolated from the Salt Lake of Chott Ech Chergui (El-Bayadh, Algeria), was found to produce an extracellular thermostable serine protease (SPLS). The highest level of protease activity detected after 6 days of incubation at 50°C was 44,600 U/mL. SPLS was purified after heat treatment for 5 min at 80°C, subjected to ammonium sulfate fractionation (80%), and Sephacryl S-200 High Resolution (HR) column purification. The purified enzyme consists of a single protein with an approximate molecular weight of 29 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence of the 24 N-terminal residues of SPLS was highly similar to those of Thermoactinomycetaceae proteases. Furthermore, the complete inhibition by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DIFP) indicates that SPLS is a member of the serine protease family. The enzyme exhibits optimal activity at pH 9 and 70°C. The thermostability of SPLS increased when 1 mM of Ca2+ was added, with a half-life of 8 h at 80°C and 3 h at 90°C. Its catalytic activity was superior to that of SPSM from Streptomyces mutabilis strain TN-X30, PREFERENZ P300, and PROTEASE Type XIV. Interestingly, SPLS demonstrated high compatibility with ISIS and Maison Det, serving as solid and liquid laundry detergents, respectively. Furthermore, performance evaluations revealed that SPLS effectively removes blood stains. Overall, SPLS exhibited interesting biochemical features, suggesting its potential use as a cleaning bio-additive in detergent formulations.
... Specifically, 8% Tris-Glycine SDS-PAGE gels were used for full-length BNT1 proteins, and 12% gels were used for the other constructs. Protein concentrations were determined using the Bradford assay (Bradford, 1976). The primary antibodies used were: mouse anti-GFP (Covance MMS-118P, 1:5000), mouse anti-GFP (Roche #10744900, 1:3000), and rabbit anti-PsbA (Agrisera AS05084, 1:10000). ...
Precise localization and trafficking of plant immune receptors are critical for their function. We identify the TNL‐class nucleotide‐binding leucine‐rich repeat receptor (NLR) BURNOUT1 (BNT1) from Arabidopsis thaliana as localized to plastids, key organelles for plant immunity. Alternative transcription start site usage generates two isoforms of BNT1: BNT1.2, which is targeted to the plastid envelope via an N‐terminal signal‐anchored mechanism, and BNT1.1, which resides in the cytoplasm. Moreover, BNT1.2 is predominantly expressed in epidermal cells, where it localizes to the so‐called sensory plastids. Functional analysis revealed that bnt1 mutants exhibit compromised PAMP‐triggered immunity (PTI) responses, including impaired callose deposition and reduced flg22‐induced resistance to Pseudomonas syringae pv. tomato, while flg22‐induced apoplastic reactive oxygen species production remains unaffected. Notably, only the plastid‐localized BNT1.2 isoform is required for these PTI responses. Our findings reveal a role for NLRs in regulating PTI responses from plastids and highlight these organelles as key hubs for signal(s) integration during plant–pathogen interactions.
... One unit of peroxidase activity is defined as the amount of enzyme that yielded one micromole (1 µmol) of product per min in one mL of assay mixture under the specified conditions. Protein concentration was quantified in both the crude and purified peroxidase by the method of Bradford [26] using bovine serum albumin as standard. The peroxidase activities were estimated in both the crude supernatant and purified enzyme with soluble protein concentration and the specific activity was thereafter calculated. ...
The kinetics of substrate and inhibitor binding to purified peroxidase from rhizome of turmeric, as well as their possible interactions with the enzyme, were investigated employing in vitro and molecular docking techniques, respectively. This was with the view to providing information on the catalytic mechanism of the enzyme with substrate and inhibitors for various applications. The crude enzyme was purified in single step purification using aqueous two-phase partitioning system (ATPS). Lineweaver–Burk plots of initial velocity data at fixed and varying concentrations of the two substrates, catechol and hydrogen peroxide, showed linear patterns with intersection on the x-axis in the third quadrant suggesting sequential ordered bi bi mechanism of substrate addition to the peroxidase. The real kinetic constants − Kmcatechol and KmH2O2 estimated from the secondary replots for the purified peroxidase from turmeric were 168 ± 2.0 mM and 87.4 ± 1.2 mM respectively. The Vmax obtained for the purified enzyme was 68,965 ± 50 units/mg protein. These led to first-order rate constant, kcat/Km of 0.49 × 106 M−1 s−1. All the inhibitors had inhibitory effect on the activity of ClP at varying concentrations. The inhibition constant (Ki) values for the inhibitors at increasing order are 0.4 mM for cysteine, 4.9 mM for ascorbic acid, 5 mM for citric acid and 9 mM for EDTA. Cysteine was the most potent inhibitor. From the docking simulation, the calculated docking score of the binding energy for ascorbic acid, citric acid, cysteine and EDTA were −8.988, −4.147, −3.361 and −2.206 kcal/mol respectively. The lower binding energy value of the inhibitor represents the higher affinity to the receptor protein. The binding interaction of the purified enzyme showed that ascorbic acid, citric acid and EDTA have 2 hydrogen bonds formed respectively while cysteine had 4 hydrogen bonds. The combination of kinetic and inhibition properties makes the enzyme a successful candidate to be employed for various applications in industrial and biotechnological processes.
... Chlorophyll a and chlorophyll b concentrations were determined using the method described by Jeffrey and Humphrey (1975). For crude protein content, the method described by Bradford (1976) was used, with bovine serum albumin as the standard. Total soluble carbohydrate was measured by the anthrone reaction with glucose as the standard (Yemm & Willis, 1954). ...
This study investigates the influence of fluctuating emersion and submersion on growth and biochemical composition of Ulva australis to elucidate their mechanisms of environmental stress adaptation. Thalli of U. australis were grown under laboratory conditions with varying emersion durations i.e. 0.5 h, 1.0 h, 2.0 h, and 5.0 h every 12 h. Biochemical composition was analyzed at final stages of thalli growth focusing on total solute carbohydrate and osmolyte content. The results showed that mild emersions (0.5 h, 1.0 h, and 2.0 h every 12 h) have significantly higher growth rate and total solute carbohydrates contents, compared to thalli without emersion, while during 5.0 h emersion, growth rate was significantly lower than those without emersion. Thalli with 5 h emersion had higher osmolyte content compared to the control (P<0.05). Under mild emersion, algae show increased total solute carbohydrate content, indicating enhanced energy storage and maintenance of metabolic processes. While increased osmolyte contents act as osmoprotectant under severe emersion to prevent cellular damage from desiccation. These observed biochemical changes provide insights into the mechanisms of stress adaptation in marine macroalgae, which could have broader implications for understanding and managing coastal ecosystems under changing environmental conditions.
... We measured the protein contents in the suspension using a test kit (Parsazmoon, Iran) and the colourimetric analytical techniques of Bradford [14]. We used bovine serum albumin (BSA) to create a protein measuring standard. ...
Due to the low-fat content and its richness in fatty acids, especially the saturated ones, and lower cholesterol levels than mutton and beef, many consumers prefer broiler chicken meat. However, the refrigerated storage may lead to chemical alterations and microbial proliferation in the meat product, generating metabolites that induce physical and chemical quality changes in the chicken meat. Thus, the study focuses on chicken meat's oxidation of lipids and proteins, microbial degradation, and physicochemical attributes during refrigeration. The study involves 30 forty-two-day-old ROSS broiler chickens at a commercial plant in Erbil, Kurdistan region of northern Iraq. After slaughtering the bids, the extracted and segmented chicken breast muscle samples into three portions: 1, 3, and 5 days postmortem after bleeding and evisceration. After a designated five-day storage period, the extracted all Pectoralis major muscle samples from their packages and sectioned them into various portions for microbiological analysis and meat quality assessments. In this study, There were increased activities of microbes, including lactic acid bacteria, Enterobacteriaceae, pseudomonas species, and total aerobic counts (P<0.05) as the days of ageing went up. However, the total bacterial count in groups was within an acceptable range. The thiobarbituric acid reactive substances, carbonyl, and free thiol contents changed significantly as the meat ages for 1, 3, and 5 days. Similarly, the ageing and refrigerated storage of broiler chicken meat affected colour, muscle pH, water holding capacity, drip loss, cooking loss, and myosin heavy chains and actin Improved refrigerated storage and meat ageing are vital to ensuring better meat quality, safety, and shelf life.
... The content of soluble protein in needles was determined according to M. M. Bradford (1976) using Coomassie Brilliant Blue G-250 dye. The optical density of the colored solution was measured on a KFK-3-01-ZOMZ photometer at a wavelength of 595 nm. ...
The research was aimed at analyzing the peculiarities of water exchange in Pinus pallasiana D. Don needles from the anti-erosion plantation on the slope and in the thalweg of the Viyskovyi ravine under different forest growth conditions. The ravine is located in the Dnipropetrovsk region and belongs to the southern geographical variant of ravine forests. The studied plants grew at three experimental sites of man-made plantation: in the thalweg (forest growth conditions – mesophilic, fresh, CL 2 ), in the middle part of the slope of the southern exposure (mesoxerophilic, somewhat dry, or semi-arid, CL 1 ), and on the upper part of this slope (xerophilic, arid, CL 0–1 ). The course of the daily intensity of transpiration, the average daily amount of midday transpiration, the humidity of the needles, its water deficit, and the water-holding capacity of the needles were studied. The research was conducted from May to September.
The curve of the daily intensity of transpiration had differences in different versions, but it had the most smoothed character in May. The maximum values of the intensity of transpiration and the amplitude of their changes were observed in July and August. During the study, the plants of the thalweg evaporated water most intensively and the plants of the upper part of the slope evaporated it least intensively. In the morning and evening, the values of this physiological process differed little in plants growing under different forest growth conditions. A significant difference was recorded at noon. The moisture content in needles in all areas was maximum in May and minimum in August, which is caused by the increase in soil dryness. During the experiment, the difference was the largest in mesophilic plant areas and smallest in xerophilic plant areas. The midday water deficit in the needles of thalweg plants was the lowest in May, but slightly increased during the summer months. In more arid conditions, compared to plants normally supplied with water, the water deficit was more pronounced, despite the decrease in the intensity of transpiration. Its maximum values in all variants were noted in August. In all variants of the experiment, a high water-holding capacity of needles was found, especially in July and August. Water loss was the highest for thalweg plants and the lowest in dry areas. The plants of xerophilic habitats have adaptations such as a decrease in the intensity of transpiration in hot hours, a shift of the maxima in their daytime course to the morning and later periods compared to plants of normally watered areas, and an increase in the water-holding capacity of the needles.
... The amount of enzyme required to degrade 1 µM min -1 of hydrogen peroxide was one catalase unit. The evaluated enzymes were expressed in enzyme units per milligram of protein (U mg -1 ) (Bradford 1976). ...
Seed germination is a critical part of plant emergence and establishment in the field, and cold stress can affect the emergence potential and seedling formation. The present study aimed to determine the differences in the plant antioxidant system among common bean seeds with contrasting vigor and assess the relationship between seed lot vigor and antioxidant system. Six seed lots of common bean genotypes (BAF42, BAF44 and BAF55) with contrasting vigor levels (high and low vigor) were submitted to germination and cold tests. Lipid peroxidation, hydrogen peroxide, catalase, guaiacol peroxidase and ascorbate peroxidase were evaluated after cold stress. The high-vigor seeds were better able to overcome the cold stress, with the catalase enzyme activity more associated with vigor. Low-vigor seeds are more susceptible to stress, with high physiological damage in seedlings, due to the greater hydrogen peroxide accumulation in the embryonic axis. In order to maintain metabolic homeostasis, the low-vigor seeds increase the production of antioxidant enzymes in the seedlings, thus exhibiting a greater antioxidant enzymatic activity and hydrogen peroxide accumulation in response to cold stress.
KEYWORDS:
Phaseolus vulgaris L.; antioxidant system; physiological seed quality; seedling performance
... The measurement of absorbance took place at 628 nm. The Bradford technique was used to calculate the protein content 52 . ...
In this work, an azo dye ligand of 2,6-dichloroaniline with nitroxoline (CPAQ), and its Zn(II), Cu(II), Cd(II), Ni(II) and Co(II) complexes have been synthesized. The structures of the synthesized compounds have been elucidated applying analytical and spectral tools. According to these results, all complexes proved to have a tetrahedral structure, except Ni(II) complex, which has an octahedral geometry. Analytical results also inferred the formation of Co(II) and Ni(II) complexes in the molar ratio 1M:1L and the remaining complexes in 1M:2L molar ratio. For further insight into the complexes’ geometry, bond lengths, bond angles, and electronic characteristics with respect to the organic ligand were assigned in addition to DFT calculations. HOMO and LOMO calculations show the Co(II) complex is more reactive. The interactions of the target compounds with the Mus musculus ADA enzyme structure (PDB ID: 1a4m) were estimated by applying molecular docking studies. The inhibitory effect of the synthesized compounds on adenosine deaminase enzyme (ADA) activity was tested in-vitro, showing the Co(II) to be the most active.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-06518-4.
... The homogenates underwent centrifugation at 10,500 g for 20 min at 4 °C. Subsequently, protein concentrations were determined in the medium and cell lysate using the Bradford method as previously described 36 . Anti-STAT-3, anti-p-STAT3, anti-EGFR, anti-p-EGFR were used. ...
Benign prostatic hyperplasia (BPH) is a prevalent progressive age-related disorder in men, yet its etiopathophysiology remains poorly understood. Current treatments like finasteride (Fin) have limited long-term efficacy, necessitating alternative therapies. Hydroxychloroquine (HCQ), a safe antimalarial agent, possesses anti-inflammatory, immunomodulatory, and antiproliferative activities, however, its therapeutic effect in BPH has not been investigated. Accordingly, we examined its therapeutic potential and underlying mechanisms, alone or combined with Fin, in testosterone-induced BPH in rats. In BPH-induced rats, HCQ markedly reduced prostate weight and index, and PSA, testosterone, dihydrotestosterone, pro-inflammatory cytokines (TNF-α, κ and IL-6), and the transcription factor “NF-κB” levels, while improving histological abnormalities in epithelial and stromal tissues. HCQ reduced the mRNA expression of AR and ERK1/2, and decreased the protein levels of EGFR and STAT3. Additionally, HCQ increased the mRNA expression of FOXO1 and promoted apoptosis through both intrinsic and TRAIL-mediated pathways. This was evidenced by the upregulation of pro-apoptotic Bax and the downregulation of anti-apoptotic Bcl-2 and Bcl-XL levels in the intrinsic pathway, as well as the reduction in mRNA expression of DR4 and DR5 in the TRAIL-mediated pathway. Notably, combining HCQ with Fin enhanced these effects. Molecular docking revealed HCQ’s strong interactions with androgen receptor (AR), EGFR, ERK1/2, FOXO, and TRAIL death receptors (DR4/DR5), comparable to Fin except for STAT3. Our findings suggest that HCQ modulates BPH progression by targeting STAT3/FOXO1/TRAIL and EGFR/ERK/AR pathways, offering a promising therapeutic strategy for BPH, either alone or in combination with Fin.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-04267-y.
... Tissue samples were homogenized in 50 mM KH 2 PO 4 / K 2 HPO 4 , pH7.8, and then centrifuged for 15 at 12.000g min at 4°C. The Bradford method [42] was used in order to estimate the tissue protein concentration. Guaiacol peroxidase GPOX activity was measured at 470 nm according to Anderson et al. [43]. ...
This study was conducted in order to test the effect of seed pretreatment or exogenous application through the rooting medium of 0.1 mM Salicylic Acid (SA) and 0.1 mM hydrogen peroxide (H2O2) on growth, nutritional behavior and some biochemical parameters (photosynthetic pigments, gas exchange parameters, oxidative stress indicators and antioxidant enzymes activities) of lentil plants (Lens culinaris) under 75 mM salt stress. Our results demonstrated that salt stress noticeably reduced shoot and root DWs by 39.01 and 42.81%, respectively, as compared to controls. This reduction was associated with a significant decrease in all photosynthetic parameters, including Chlorophyll (Chl) and carotenoid (Car), net assimilation of photosynthesis (A), stomatal conductance (gs), transpiration (E) and internal CO2 level (Ci), an accumulation of Na⁺ and Cl⁻ and a decrease of K⁺ and Ca²⁺ concentrations in plant shoots and roots. In addition, relative to control plants, salt stress remarkably increased the malondialdehyde MDA and H2O2 contents especially in roots and increased GPOX and SOD activities, especially in plant shoots. Both methods of SA and H2O2 application recovered the plant growth, enhanced shoot and root DWs (increase of 67.65 and 82.36% in shoots and roots, respectively, as compared to salt-stressed plants) and increased all parameters that were reduced by NaCl treatment. Nevertheless, the most prominent effects of SA and H2O2 on plant growth were obtained with the seed priming method. Thus, SA and H2O2 applications, especially the H2O2 seed priming method, induced the antioxidant system, improved the membrane stability and ameliorated the gas exchange parameters. As compared to salt plant stressed, Na⁺ and Cl⁻ contents were significantly decreased and K⁺ and Ca²⁺ were significantly increased in shoots and roots following SA and H2O2 applications, especially with the H2O2 seed priming method. Similarly, this method was more efficient in alleviating the adverse effects of salt stress on all photosynthetic pigment contents and measured gas exchange parameters. Compared to salt stressed plants, it significantly decreased the H2O2 and MDA contents and further stimulated GPOX and SOD activities. Our results indicated that the seed priming method, particularly with H2O2, could be recommended for obtaining better growth of lentil seedlings under salt-affected soil conditions.
... The NJ method reduces overall branch length by iteratively grouping taxa depending on pairwise distance measures. The phylogenetic tree was generated using MEGA's Tree Explorer, which offers interactive capabilities for analyzing tree topology and [27], with bovine serum albumin (BSA) acting as the protein standard. ...
The present study investigates the spoilage indices and physicochemical parameters of Longissimus lumborum (LL) and Semitendinosus (ST) muscles of goats under super-chilling one degree Celsius (1 °C) conditions compared to refrigerated degree Celsius (4 °C) and frozen degree Celsius (-18 °C) samples over a seven-day post-mortem ageing period. Fifteen local bucks obtained from a commercial farm were used for the study. Results show that super-chilling and frozen storage methods significantly reduced microbial spoilage, and the structure of the bacterial community in goats’ muscles altered over an extended period, contrasting with meat stored under refrigerated conditions. Primarily, the dominant bacterial species identified in muscle samples of goats at the genus level were Pseudomonas, Myroides, and Brochothrix. Pseudomonas has been mainly identified as the bacterium responsible for spoilage under refrigerated conditions (36.23% for longissimus lumborum muscle and 25.45% for semitendinosus muscle). However, the relative abundance of Pseudomonas was significantly reduced in both frozen and super-chilled longissimus lumborum (9.334% and 8.229%, respectively) and semitendinosus (7.098% and 6.999%, respectively) muscle samples, and these results revealed that the low temperature effectively inhibited the growth of Pseudomonas. Muscle samples stored under super-chilled and frozen conditions recorded a slower lipid and protein oxidation rate following ageing. Super-chilling effectively reduced deterioration associated with ice crystal formation and enzyme-mediated changes, which are significantly different from freezing and refrigerated storage methods. The quality attributes of LL and ST were minimally impacted. Super-chilling is practical and can be used for storage as it ensures the quality preservation of meat.
Salinity and heavy metal stress significantly reduce agricultural productivity in arable lands, particularly affecting crops like rice (Oryza sativa L.). This study aimed to evaluate the efficacy of heavy metal-tolerant plant growth-promoting rhizobacteria (HMT-PGPR) in mitigating the harmful effects of salt (NaCl), chromium (Cr), and combined NaCl + Cr stress on rice plants. Two pre-isolated and well-characterized heavy metal-tolerant epiphytic (Ochrobactrum pseudogrignonense strain P14) and endophytic (Arthrobacter woluwensis strain M1R2) PGPR were tested. The LSD test (p ≤ 0.05) was used to assess the statistical significance between treatment means. Stresses caused by NaCl, Cr, and their combination were found to impair plant growth and biomass accumulation through mechanisms, including osmotic stress, oxidative damage, ionic imbalance, reduced photosynthetic pigment, lowered relative water content, and compromised antioxidant defense systems. Conversely, inoculation with HMT-PGPR alleviated these adverse effects by reducing oxidative stress indicators, including malondialdehyde (MDA), hydrogen peroxide (H2O2) content and electrolyte leakage (EL) and enhancing plant growth, osmolyte synthesis, and enzymatic antioxidant activity under single- and dual-stress conditions. The application of HMT-PGPR notably restricted Na+ and Cr6+ uptake, with an endophytic A. woluwensis M1R2 demonstrating superior performance in reducing Cr6+ translocation (38%) and bioaccumulation (42%) in rice under dual stress. The findings suggest that A. woluwensis effectively mitigates combined salinity and chromium stress by maintaining ion homeostasis and improving the plant’s antioxidant defenses.
Retinal ribbon synapses are continuously active chemical synapses. The eponymous synaptic ribbon is anchored to the active zone neurotransmitter release sites of ribbon synapses, recruits synaptic vesicles and guides ribbon-associated synaptic vesicles to the release sites. RIBEYE is the major protein component of synaptic ribbons. But likely, additional proteins contribute to ribbon synapse function. The synaptic ribbon of photoreceptor synapses is embedded into a highly polarized microtubule cytoskeleton. Interestingly, proteins of the photoreceptor primary cilium, such as NPHP4 and other ciliary proteins, including KIF3A, were shown to be localized to photoreceptor synaptic ribbons. Previous studies demonstrated that the microtubule motor protein KIF13B catalyzes secretory vesicle transport to the plus ends of microtubules and identified an interaction of KIF13B with NPHP4 at primary cilia. However, the localization of KIF13B, a kinesin-3 family motor protein, in the retina is still unknown. In the present study, we used two different antibodies against KIF13B and high-resolution confocal microscopy, super-resolution structured illumination microscopy (SR-SIM), and post-embedding immunogold electron microscopy to determine the localization of KIF13B in retinal photoreceptors. Apart from its localization at the primary photoreceptor cilium, we found a strong enrichment of KIF13B at photoreceptor synaptic ribbons. The synaptic ribbon is needed for the synaptic enrichment of KIF13B as shown by analyses of synaptic ribbon-deficient RIBEYE knockout mice. These findings suggest that KIF13B performs vesicle trafficking functions at the photoreceptor synaptic ribbon complex at the interface between the synaptic ribbon and the presynaptic microtubule transport system.
This study investigated the dynamics and composition of microbial communities within the bioreactors of a two-stage anaerobic system employed for the bioconversion of corn steep liquor, a food processing byproduct, into hydrogen and methane. The high organic matter content of such wastes positions them as valuable substrates for biotechnological applications. The two-stage anaerobic digestion (AD) process was compartmentalized into a hydrogen-producing bioreactor (3 dm3) and a methane-producing bioreactor (15 dm3), each harboring distinct microbial consortia. The system yielded a maximal hydrogen production of 1.02 L/day and a peak methane production of 24.1 L/day with substrate corn steep liquor and cattle manure in a ratio 1:1. Microbial consortia were recognized as critical drivers of AD performance and biofuel yield. This research demonstrated the efficacy of a two-stage approach, segregating the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis) phases, for optimized energy recovery from the co-digestion of corn steep liquor and cattle manure under controlled conditions. Metagenomic sequencing and a subsequent bioinformatics analysis were utilized to characterize the microbial diversity within each bioreactors. These findings contribute to a deeper understanding of the microbial ecology of AD and hold the potential for broader applications in waste-to-energy bioconversion.
Secreted bacteriolytic proteases L1 and L5 of the Gram-negative bacterium Lysobacter capsici XL hydrolyze peptide bridges in bacterial peptidoglycans. Such specificity of action determines the prospects of these enzymes for medicine with the view of creating new antimicrobial drugs to combat antibiotic-resistant strains of pathogens. This research concerns the development of successful expression systems for producing active enzymes L1 and L5 in sufficient amounts for comprehensive studies. Based on L. capsici XL strains with deletions in the alpA (enzyme L1) and alpB (enzyme L5) genes and the constructed expression vectors pBBR1-MCS5 PT5–alpA and pBBR1-MCS5 PT5–alpB, we obtained expression strains L. capsici PT5–alpA and L. capsici PT5–alpB, respectively. The yields of enzymes L1 and L5 in the developed strains increased by 4 and 137 times, respectively, as compared to the wild-type strain. The cultivation of the expression strains was successfully scaled up under non-selective conditions in a 10-L bioreactor. After fermentation, the yields of enzymes L1 and L5 were 35.48 mg/L and 57.11 mg/L, respectively. The developed homologous expression systems of bacteriolytic proteases L1 and L5 have biotechnological value as compared to those obtained by us earlier based on heterologous expression systems, which have lower yields and labor-intensive purification schemes.
The aims of the present study are to examine how lettuce plants (Lactuca sativa L.) responded to salinity stress in relation to four treatment groups: exogenous zinc oxide nanoparticles (ZnO-NPs) group, control group, sodium chloride (NaCl) group, and sodium chloride + exogenous zinc oxide nanoparticles (NaCl + ZnO-NPs group).
Cos Lettuce (Thai – MGS1376) was utilized as the experimental material. Four different treatments, such as zinc oxide (ZnO), exogenous zinc oxide nanoparticles (ZnO-NPs), NaCl, and sodium chloride + exogenous zinc oxide nanoparticles (NaCl + ZnO nanoparticles) were used.
The findings revealed that exogenous ZnO-NPs might increase growth and biomass of plants in salinity-stressed environments. After applying various ZnO-NPs, lettuce leaves potassium ion (K+) concentration and potassium to sodium ion (K+/Na+) ratio increased. Significant changes were found in proline content, malondialdehyde (MDA) content, and superoxide anion (O2−) generation between ZnO-NPs and control groups (p ≤ 0.05). There were no significant differences between the ZnO-NPs and control groups in terms of soluble sugar content, superoxide radical production rate, hydrogen peroxide (H2O2) content, or ascorbic acid content, implying that oxidative damage to the leaves was reduced. ZnO-NPs enhanced shoot and root dry weight, fresh weight, and leaf area, alongside increased chlorophyll levels.
The findings are implying ZnO-NPs’ potential for boosting lettuce growth in saline conditions. The ZnO nanoparticles can be a helpful ecologically friendly treatment to improve plant tolerance and antioxidant activity, hence reducing salt stress-related damage.
Biorefinery is gaining attention as a promising approach to valorize natural resources and promote a circular bioeconomy. This study aimed to recover high-value molecules, such as xanthophylls and polar lipids with nutraceutical applications, through enzymatic pretreatment and sequential pressurized liquid extraction (PLEseq), by reusing the residual biomass of Nannochloropsis gaditana after each processing step. Remarkably, pure glycolipids (102.95 ± 1.10 mg g−1 dry weight) were obtained immediately after enzymatic pretreatment, facilitating their easy recovery. Furthermore, two alternative sequential extraction processes were successfully developed, using ethanol and water as green solvents at varying temperatures and in different orders. The most effective PLEseq conditions yielded up to 48 mg mL−1 of carbohydrates using water at 50 °C, and up to 44 mg mL−1 of proteins via subcritical water extraction at 100 °C, prior to conventional lipid extraction with ethanol to produce various concentrated extracts. In the inverted PLEseq process—starting with ethanol extraction followed by successive water washes—isolated and purified fractions of lutein and astaxanthin were obtained, contributing to the complete depletion of the residual biomass. Overall, the development of an integrated and sequential biorefinery protocol that enables the extraction of multiple high-value compounds holds significant potential for application in the food industry.
Brewery’s spent grain (BSG) consists of the largest by-product by volume in the beer production sector and offers potential for both bio-composite material production, high-added-value molecular extraction and bioenergy recovery. Aiming at exploring the ideal biorefinery approach for this agro-industrial residual, the present study experimentally investigated several methodologies to enhance the reuse of BSG and proposed a scheme of biorefinery focused on it. According to it, BSGs were firstly tested to produce high-added-value byproducts, such as protein hydrolysates and for the extraction of lignin via ionic liquids-based methods. The residuals were then used for biogas/biomethane production via anaerobic codigestion. The different matrices were rearranged in varying mixtures, aiming at ensuring high availability of nutrients for methanogens, thus achieving higher energy production than what achievable with untreated BSG. For the scope, further agro-industrial wastes were considered. The resulted digestate was finally composted. Untreated BSGs were also directly tested as fillers for bio-composite material production (in a mixture with PHB). Different concentrations were tested and the mechanical properties of each sample were compared with those of pure PHB. Disintegration tests were finally carried out to measure the improved biodegradability of the produced bio-composite material.
The objective of the present work was to elucidate peculiar features of protein composition of the lacrimal fluid in children and adolescents with high progressive myopia. Thirty five samples of the lacrimal fluid obtained from35 children and adolescents at the age from 10 to 17 (mean 13,1 ± 2,2) years with myopia (5,0 – 20,5 D, mean9,5 ± 3,7 D) developing at the annual progression rate of 0,98 ± 0,8 D. were available for the analysis. Nineteenpatients had various forms of peripheral vitreochorioretinal dystrophy. None of the patients included in the studyused contact lenses to correct myopia. Nine samples of lacrimal fluid were taken from children and adolescents ofthe same age with emmetropy without changes in the eye fundus. The samples of lacrimal fluid from the patientsof the two groups were significantly different in that they contained much less total protein in the children withhigh progressive myopia compared with the control subjects. At the same time, the relative amount of lactoferrin(one of the major proteins of lacrimal fluid exhibiting antioxidative and metal-chelating activities) in the formergroup was significantly higher. The results of the study on the one hand confirm the pathogenetic role of thedisorders in the antioxidative protective system of the eye tissues and media and on the other hand give reasonto consider the reduction of the total protein content and the rise in the lactoferrin fraction in the lacrimal fluid asthe diagnostic signs of progressive development of the myopic process.
The limited efficacy of antipsychotics in treating the negative and cognitive symptoms of schizophrenia has prompted the exploration of adjuvant therapies. Several drugs developed for other indications—including caffeine, metformin, and furosemide—have shown procognitive potential. This study evaluated the effects of these agents on behavioral parameters using the reward-based Ambitus test, and on the cerebral D2 dopamine receptor (D2R) expression and binding. The drugs were administered individually and in combination in a schizophrenia-like triple-hit animal model (Lisket rats), derived from the Long Evans (LE) strain. Lisket rats received 14 days of drug treatment via drinking water; water-drinking LE rats served as the controls. The Ambitus test was conducted before treatment and on days 11–14. Caffeine enhanced activity without affecting learning or memory. Metformin and furosemide reduced exploratory behavior but improved reference memory; these effects were inhibited by caffeine co-administration. Although no statistically significant behavioral differences were found compared to water-treated Lisket rats, a trend toward reduced exploratory visits was observed in the triple-combination group. Lisket rats exhibited moderately reduced D2R binding in the cortex and increased binding in the hippocampus. Caffeine alone and in combination enhanced hippocampal D2R binding, while furosemide increased cortical D2R expression. This study is the first to highlight the behavioral and molecular effects of these non-antipsychotic agents in a schizophrenia model, supporting their potential for adjunctive use.
Soil salinity degrades land worldwide, especially in semi-arid and arid regions. It arises naturally because of limited rainfall and the deposition of soluble salts at the soil surface, exacerbated by human activities such as more flood irrigation, excessive use of fertilizers, and inadequate drainage. The current study was conducted to assess the synergistic effect of melatonin (ML) and strigolactone (SL) on maize seedlings to ameliorate the harmful effects of salinity. The 250 µM ML and 25 µM SL were applied alone or in combination on maize seedlings grown under salinity stress (100 mM NaCl) and normal conditions (control, Ck). Salinity stress reduced growth and physiological parameters, but ML and SL, especially in combination, significantly improved these traits. Salinity stress significantly (p < 0.05) augmented H2O2, malonaldehyde (MDA) contents, electrolyte leakage, and Na+ concentration in maize seedlings as compared to Ck. Nevertheless, the combined application of ML and SL alleviated the oxidative stress caused by salinity stress by increasing the activities of enzymatic antioxidants (superoxide dismutase, peroxidase, ascorbate peroxidase, and catalase) and decreasing the levels of H2O2, MDA, electrolytes leakage, and Na+ concentration. In addition, free proline, soluble protein, total soluble sugar, total phenolics, and K+ contents were remarkably stimulated by applying ML + SL under saline conditions. This study demonstrates that ML and SL synergistically enhance maize resilience to salinity stress by strengthening antioxidant defenses, improving osmotic balance, and maintaining ion homeostasis. These findings suggest that exogenous ML and SL application is a promising strategy for cultivating maize in salt-affected areas.
With the continuous expansion of oyster farming scale, disease has become one of the main obstacles to restricting the development of oyster farming. In the present study, 20 bacterial strains were identified from Crassostrea gigas with pustulosis, among which Pseudoalteromonas aliena emerged as the predominant strain, characterized by its rod-shaped morphology and possession of flagella. P. aliena exhibited α-hemolytic activity at 28°C and displayed high susceptibility to all 20 chemotherapeutic agents tested. After P. aliena infection, the oyster mortality rate increased. The gills were swollen and eroded, and the mantle was green with pustules after P. aliena infection. The gill filaments exhibited swelling and necrotic cells, and the mantle showed a loose histological structure with cavities and disruption of epithelial cells. The extracellular products (ECPs) from P. aliena had urease, protease, and amylase activities. The potential virulence proteins identified from ECPs were GroL, ClpB, and HtpG proteins. After injection with ECPs, there was an increase in the oyster mortality rate, and the observed symptoms in gill filaments and mantle were consistent with those observed after P. aliena infection. In addition, the mRNA expressions of inflammation- and programmed cell death-related genes were significantly upregulated in gills and mantle. The relative abundances of Vibrio , Arcobacter, and Pseudoalteromonas also exhibited a significant increase in the gills and mantle. The results demonstrated that P. aliena was the pathogenic bacterium for oysters, and its pathogenicity mechanism was systematically clarified, which provided valuable insights for the prevention and control of bacterial disease in oysters.
IMPORTANCE
Disease has currently emerged as one of the principal impediments to restricting the development of the oyster breeding industry. In the present study, Pseudoalteromonas aliena was identified from Crassostrea gigas with pustulosis. After P. aliena infection, the oyster mortality rate increased. The gills were swollen and eroded, and the mantle was green with pustules. Extracellular products (ECPs) from P. aliena had urease, protease, and amylase activities. The potential virulence proteins identified from ECPs were GroL, ClpB, and HtpG proteins. After injection with ECPs, the oyster mortality rate increased. The mRNA expressions of inflammation- and programmed cell death-related genes in gills and mantle increased significantly, and the relative abundances of Vibrio , Arcobacter, and Pseudoalteromonas exhibited a significant increase after P. aliena infection. The results demonstrated that P. aliena was the pathogenic bacterium for oysters, and its pathogenicity mechanism was systematically clarified, which provided valuable insights for the prevention and control of bacterial disease in oysters.
Cydalima perspectalis (Walker, 1859) (Lepidoptera: Crambidae) larvae feed on Buxus . It is considered to be the most critical pest of boxwood trees. This study investigated whether different strains of Trichoderma harzianum had an effect on the biocontrol of larvae feeding on boxwood leaves whose nitrogen content was varied by fertilisation. Larvae were collected while feeding on boxwood seedlings in Rize parks and gardens in June 2021. In addition, G1 (no fertilisation), G2 (1.55%), and G5 (1.67%) leaves with different nitrogen concentrations obtained by nitrogen fertilisation were also used as food. As biocontrol agents, ID11D and YP1A strains of T. harzianum were applied in three doses: 50, 100, and 200 μL per water. In total, 21 different groups were created. The nutritional indices of the larvae belonging to the different groups were calculated. In addition, the activities of phenoloxidase, superoxide dismutase, catalase, and glutathione peroxidase activities were measured by taking haemolymph samples. In both strains, the enzyme activities increased with the dose applied. However, it was found that the enzyme activities of the ID11D strain applied were higher than those of the YP1A strain. It can be said that the ID11D strain is effective in controlling C. perspectalis larvae feeding on fertilised boxwood and the YP1A strain is effective in controlling larvae feeding on unfertilised boxwood.
Biocatalytic approaches, leveraging enzymatic and molecular mechanisms, offer sustainable solutions for degrading environmental pollutants. This review explores the role of microbial enzyme transforming persistent contaminants such as polycyclic aromatic hydrocarbons, dyes, and endocrine disruptors into less harmful compounds. The specificity and efficiency of these enzymes make them effective in various environmental matrices. Advancements in enzyme immobilization techniques have enhanced their stability and reusability, addressing challenges related to operational conditions and cost-effectiveness. Furthermore, molecular strategies, including protein engineering and metagenomics, have facilitated the discovery and optimization of novel enzymes with improved degradation capabilities. This review underscores the potential of integrating enzymatic and molecular tools in bioremediation efforts, aiming for efficient and eco-friendly pollutant degradation strategies.
The effects of a fungal elicitor from Trichothecium roseum on signal pathways of salicylic acid (SA), jasmonic acid (JA), and Ca2+ in potato tubers were investigated. The results showed that fungal elicitor treatment effectively inhibited the lesion diameter of Fusarium sulphureum in vivo, which was 17.5% lower than that of the control. In addition, fungal elicitor treatment triggered an increase in O2− production and H2O2 content. The fungal elicitor enhanced the activities and gene expression levels of isochorismate synthase (ICS), phenylalanine ammonia lyase (PAL), allene oxide cyclase (AOC), allene oxide synthase (AOS), lipoxygenase (LOX), and Ca2+-ATPase. Furthermore, the fungal elicitor promoted an increase in calmodulin (CaM) content. Protective enzymes (dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO), chitinase (CHI), and β-1,3-glucanase (Glu)) and disease-resistance-related genes (PR1, PR2, and PDF1.2) were induced to be upregulated by elicitor treatment. These results indicated that the fungal elicitor induced disease resistance by accelerating the accumulation of reactive oxygen species (ROS), activating SA, JA, and Ca2+ signaling, and upregulating resistance genes. The results of this study revealed the molecular mechanism of fungal elicitor-induced resistance in the potato, which provides a theoretical basis for the mining of new, safe, and efficient elicitor-sourced antifungal agents and is of great importance for the effective control of potato dry rot disease.
Biocatalytic approaches, leveraging enzymatic and molecular mechanisms, offer sustainable solutions for degrading environmental pollutants. This review explores the role of microbial enzyme transforming persistent contaminants such as polycyclic aromatic hydrocarbons, dyes, and endocrine disruptors into less harmful compounds. The specificity and efficiency of these enzymes make them effective in various environmental matrices. Advancements in enzyme immobilization techniques have enhanced their stability and reusability, addressing challenges related to operational conditions and cost-effectiveness. Furthermore, molecular strategies, including protein engineering and metagenomics, have facilitated the discovery and optimization of novel enzymes with improved degradation capabilities. This review underscores the potential of integrating enzymatic and molecular tools in bioremediation efforts, aiming for efficient and eco-friendly pollutant degradation strategies.
Water deficit and heat have been considered the main abiotic stresses to which forests are exposed due to global warming. Since seedling establishment is a vulnerable developmental transition that plays a critical role in forest dynamics, we carried out in vitro experiments to reveal how seedlings of the Caatinga native species Pityrocarpa moniliformis (Benth) Luckow & R.W. Jobson respond to water deficit and heat with respect to growth, water status, reserve mobilization, hydrolase activity, and metabolite partitioning, including non-structural carbohydrate availability. In the seedlings exposed to osmotic stress induced by mannitol, restricted growth was not accompanied by alterations in water status, but it was associated with delayed mobilization of starch and storage proteins in the cotyledons and decreased availability of non-structural carbohydrates in the root. By contrast, high temperature retarded the establishment of the photosynthetic apparatus and compromised the activity of hydrolytic enzymes, even though reserve mobilization has not been changed in the cotyledons. Considering the absence of mortality, it is suggested that P. moniliformis may be tolerant to osmotic and heat stress during seedling establishment. The maintenance of water status seems crucial to water deficit tolerance, while the availability of non-structural carbohydrates may play a role in heat stress tolerance.
1. Our earlier preparations of bovine chymotrypsinogen B isolated by chromatography on O-(carboxymethyl)cellulose have been further purified by O-(carboxymethyl)Sephades chromatography. the product finally obtained (yield, 25%; 400 mg per kg fresh pancreas) is entirely free from deoxyribonuclease and behaves as an homogeneous protein when subjected to free-boundary electrophoresis, ultracetrifugation and equilibrium chromatography. Chromatography on O-(carboxymethyl)-Sephadex also appears to be a good technique for the purifition of bovine deoxyribonuclease.
The ionization of the phenolic hydroxyl groups of bovine serum albumin has been studied by measuring the ultraviolet light absorption at 295 mμ as a function of pH at three temperatures. The ionization curves are steeper than theory would predict, and can be fitted with a computed curve only if the effective negative charge on the albumin molecule is assumed not to rise much above -50. The intrinsic pK for ionization at 25° lies between 10.0 and 10.3; the heat of ionization is 11.5 kcal./mole; the intrinsic entropy of ionization is -8 e.u. The heat of ionization of di-iodo phenolic groups in iodinated albumin is 7.0 kcal./mole. These figures all suggest that the phenolic OH group in native albumin may be hydrogen-bonded; however, the hydrogen bonds are certainly not involved in maintaining the native structure of the molecule, for the ionization proceeds instantaneously and reversibly.
A method has been developed to stain rapidly protein zones not only in standard but also in isoelectric focusing polyacrylamide gels. It requires no destaining in either case. The technique makes use of the fact that the G250 form of Coomassie Brilliant Blue exhibits a color change in dilute perchloric acid which is reversed when the dye becomes bound to the protein. Under the conditions used, Ampholine shows little interference. In addition, the method selectively visualizes the arginine-rich histones because of the solubility of the lysine-rich histones in PCA.
A protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B. The proteindye complex is eluted, and its absorbance determined at 630 nm. This assay is very reproducible, insensitive to variations in assay conditions, and linear from 3 to 30 μg of protein. It can be used on samples with a concentration as low as 0.75 μg/ml. There is no interference by commonly used reagents such as Tris, thiol reagents, EDTA, urea, sucrose, and many others. The color yield for a variety of proteins was determined and found to lie within ±15% of the value for bovine serum albumin which was used as standard. Of the proteins tested only insulin, which due to its low molecular weight was incompletely retained on the membrane in the filtration step, gave a low color yield, 50% of the standard.
The Pro-Milk method was compared to the official final action Udy method, 16.037-16.040, for 166 samples of raw milks from individual cows and to the official final action Kjeldahl method, 16.035, for 54 similar samples. Averages ± standard deviations of the differences were 0.019±0.017 and 0.027±0.109% protein for the 2 methods, respectively. Fat test or protein level of the samples did not affect the magnitude of the difference between the Pro-Milk and either of the other 2 methods. The Pro- Milk comparisons have also been made on samples of processed milks with similar results. A number of comparisons of the Pro-Milk method to other methods for protein in milk have been reported in the literature. The principles of the Pro-Milk method differ from the Udy method only in the type of dye used. Periodic calibration and routine control of the Pro-Milk method is required because of variability in assay of the Amido Black 10B dye.
THE quantity of protein in dilute solution is usually determined by the method of Lowry et al.1, using phenol reagent. This method is sensitive and simple, but it is applicable only to solutions containing more than 10 µg/ml. and it suffers from interference by various chemicals. For example, tris(hydroxymethyl) aminomethane can react with the reagent to develop an intense colour and in the presence of magnesium ions the phenol reagent forms a precipitate which greatly lessens the strength of the colour. Thus unless freed from these materials the values obtained under these conditions are rather unreliable. Removal of the interfering chemicals cannot be easily performed with dilute protein solutions and is inconvenient if a large number of samples is being analysed. Protein determination by ultra-violet absorbancy may also suffer from severe interference by other chemicals. The reading is affected by nucleotides or many other chemicals and the results must be corrected for them2.
The light absorption band of the peptide bond at 191–194 mμ has been used for quantitative measurement of purified peptides and proteins with the Beckman DBG spectrophotometer. In comparison to measurement of proteins at 280 mμ those conducted at 191–194 mμ are approximately 40–80 times more sensitive, depending on the protein. This is a significant advantage, especially in monitoring of effluents from chromatographic columns. Specific absorbance values of different proteins at 191–194 mμ vary less widely than those at 280 mμ and, therefore, in cases in which proteins cannot be analyzed in terms of nitrogen or protein weight, analysis by photometry at 191–194 mμ is subject to less uncertainty than that at 280 mμ. The average specific absorbance (A1cm1%) for 5 proteins was 686 with the Beckman DBG spectrophotometer. With this instrument we have observed direct proportionality between absorbance and concentration up to an absorbance of about 0.7. All anions except fluoride interfere since they absorb near 190 mμ. Therefore, the only electrolytes which can be used in appreciable concentration are those containing fluoride as anion.
Zusammenfassung Es wird die Interferenz von Natrium Ethylendiaminotetraacetat bei der Eiweissbestimmung nach der Methode vonLowry et al. und deren Verhinderung gezeigt. Die Anwesenheit von Natrium Ethylendiaminotetraacetat interferiert bei der Eiweissbestimmung nach der Methode vonLowry, da sie selbst zur Reduktion des Folin-Reagens führt. Es gelingt, diese Interferenz durch Zusatz von CaCl2 zu verhindern.
Equations for protein in sow's milk by Orange G dye binding procedures were: per cent protein = 9.184-4.526 (absorbance) at Sensitivity 1 for samples above 6.5% protein, and per cent protein = 7.317-6.061 (absorbance) at Sensitivity 2 for samples containing 3.0 to 6.5% protein. Forty-two samples of sow's milk were analyzed with a Beckman Model B spectrophotometer. Sow's milk had more protein than cow's milk, requiring 1 ml milk per 25 ml Orange G dye. Protein analyses by Orange G dye binding were 0.95 correlated with Kjeldahl estimates of protein. These equations differed from those developed for estimating protein of cow's milk by the Orange G procedure.
Glycerol interferes with the estimation of protein by both the Lowry and the biuret procedures. Color development in the presence of protein is lessened by the presence of glycerol in samples tested by either assay. In addition, with the Lowry test significant color development linearly related to glycerol concentration is obtained in the absence of protein. Problems in either assay of protein estimation in the presence of glycerol may be overcome by the use of appropriate blanks and calibration curves.
SEVERAL substances that are present in biological materials and some compounds which are added during protein purification procedures interfere with protein determinations by the biuret and Folin-phenol procedures1. For example, substances with two or more peptide bonds and ammonium salts give a positive biuret reaction and aromatic amino-acids, uric acid, guanine, and xanthine cause similar difficulties in the Folin-phenol method. Reducing agents such as 2-mercaptoethanol interfere with both procedures. The effects of these substances can be minimized if protein samples are analysed after being precipitated, centrifuged and further washed with trichloroacetic acid (TCA). It, however, the initial protein solution is very dilute and large portions must be taken for analysis, or if only a small amount of protein is to be assayed, problems arise in recovering and washing the precipitated protein free of interfering substances. Micromethods have been devised2 to surmount these problems, but for routine analysis a rapid and effective procedure is needed for removing these undesirable substances and determining protein. These requirements appear to be fulfilled in the method reported here. This method involves the analysis of protein precipitated by TCA and then separated from interfering substances by membrane filtration on `Millipore' filters.
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