Na-phosphate Buffer Research Articles

Synergic and Additive Effects of the Combined Effect of Tyrosinase Inhibitors

Background. Excessive melanin accumulation in the skin can lead to various diseases and cosmetic issues. While tyrosinase inhibitors are commonly used to reduce pigment biosynthesis, many of them are associated with significant side effects. When multiple drugs are used in combination, it can result in synergism, additive effects, or antagonism. Combining multiple tyrosinase inhibitors is considered a promising approach to minimize side effects and enhance therapeutic efficacy. Objective. This study aims to investigate the combined use of tyrosinase inhibitors to determine the nature of their interaction, whether it's synergistic or additive. Methods. We utilized tyrosinase isolated from Agaricus bisporus mushrooms. Enzyme inhibition by test compounds was assessed by measuring tyrosinase activity using tyrosine (30 min in 0.05 M Na-phosphate buffer solution, pH 6.5, 25 °C). To explore joint inhibition, compound solutions were mixed in pairs at various concentrations. The interaction was quantified using the combination index and isobolograms. Results. To determine the effect of the combined action of agents on tyrosinase activity, we examined standard inhibitors of the enzyme (kojic acid, arbutin, phenylthiourea) and our discovered compound, 3-(2-hydroxyphenylamino)-1,3-dihydro-indol-2-one. Calculations of the combination index and isobolograms for all studied combinations of standard tyrosinase inhibitors revealed additive effects in all studied cases. Simultaneous use of kojic acid or arbutin with 3-(2-hydroxyphenylamino)-1,3-dihydro-indol-2-one demonstrated a synergistic effect. However, the mixture of phenylthiourea with the indole derivative demonstrated an additive effect. Conclusions. The combined usage of tyrosinase inhibitors in various combinations displayed both additive and synergistic effects. The synergistic effect of using two inhibitors simultaneously presents significant opportunities for the development of more effective and cost-efficient treatments for hyperpigmentation by reducing the concentration of each inhibitor.

Effect of Surface Curvature on Colloidal Stability of Silver Nanoparticles with Monomolecular and Mixed Thiol Ligand Layers in the Presence of Alkali Cations.

Colloidal stability of silver nanoparticles is the critical parameter while designing colloidal colorimetric biosensors. Here, we examined colloidal stability of 11-mercaptoundecanoate-capped quasi-spherical silver nanoparticles and silver nanoplates in 0.02 M phosphate buffers with pH 8.0 containing Li+ , Na+ , K+ , or Cs+ cations. While quasi-spherical nanoparticles demonstrate a good colloidal stability in the presence of all studied cations, nanoplates aggregate in the presence of Na-phosphate buffer. The mechanism of aggregation consists in the ion-specific nanoparticle-cation bridging interaction, which is sensitive to the nanoparticle surface curvature. Increased apparent dissociation constant of carboxyl groups on the zero-curvature nanoplates' surface enhances bridging interactions and makes nanoplates colloidally unstable. Bridging interactions can be eliminated by using mixed bimolecular 11-mercaptoundecanoate-11-mercaptoundecanol surface ligand layer. Silver nanoplates with mixed ligand layer show an enhanced colloidal stability at a standard carbodiimide bioconjugation protocol.

Aggregative stability of colloidal 3D and 2D silver nanoparticles, stabilised by 11-mercaptoundecanoic acid, in the presence of singly charged cations

We studied the aggregative stability of colloidal silver quasi-spherical nanoparticles and two-dimensional nanoplates, stabilised by 11-mercaptoundecanoic acid, in the presence of phosphate buffers containing different singly charged cations (Li+ , Na+ , K+ , Cs+) and tris-HCl at pH 8.0 and concentration 0.02 mol/L which mimics the carbodiimide conjugation conditions of nanoparticles with biomolecules. Aggregation of silver nanoplates occurs in the presence of Na-phosphate buffer whereas at the same conditions the quasi-spherical nanoparticles retain colloidal stability. The difference in colloidal stability between 3D and 2D silver nanoparticles is due to the increase of the apparent acid dissociation constant on the nanoplates’ basal faces and the subsequent increase in specific bridging interactions nanoparticle – cation – nanoparticle which can be eliminated by introducing of non-ionic spacer (11-mercapto-1-undecanol) in the ligand layer. Silver nanoplates with mixed ligand layer have increased colloidal stability across the pH.

Acclimation and Characterization of Marine Cyanobacterial Strains Euryhalinema and Desertifilum for C-Phycocyanin Production.

This study involves evaluation of two native cyanobacterial strains Euryhalinema and Desertifilum isolated from a mangrove pond in Haikou (China) for their possible phycocyanin (C-PC) production. Maximal growth rate with highest chlorophyll and C-PC accumulation were observed at 28°C and 60 μmol photons m−2 s−1 photon flux density for Euryhalinema sp., while for Desertifilum sp. at 32°C and 80 μmol photons m−2 s−1. Nitrogen and iron concentration trails revealed that double strength concentration of sodium nitrate and ferric ammonium citrate in original BG11 media increased growth rate and accumulation of C-PC for both strains. Three different C-PC extraction methods were tested. The combined extraction protocol of freeze–thaw and ultrasonication markedly increased the C-PC extraction efficiency and attained the food grade purity (A 620/A 280 ratio >0.7), whereas a higher C-PC yield was found with Na-phosphate buffer. Furthermore, the clarified crude extract was used to purify C-PC by fractional ammonium sulfate [(NH₄)₂SO₄] precipitation, Sephadex G-25 gel filtration chromatography, and DEAE-sephadex ion exchange chromatography and attained analytical grade purity (A 620/A 280 ratio >3.9). Taken together, both strains showed their potential to be domesticated for valuable phycocyanin production.

IN VITRO ANTIOXIDANT ACTIVITY OF C-PHYCOCYANIN PURIFIED FROM Spirulina platensis DRY BIOMASS

C-phycocyanin (C-PC) was a phycobiliprotein found exclusively in cyanobacteria as Spirulina and exhibited a variety of pharmacological properties, which were not yet studied for any purpose in commercial applications in the pharmaceutical and cosmetic industry in Vietnam. At present, there are no other sources of natural phycocyanin than Spirulina, it is the sole efficient source of natural phycocyanin production under photoautotrophic conditions. The present research was conducted to determine an efficient extraction method from C-PC from Spirulina platensis dry biomass. Antioxidant activity of purified C-PC was investigated. Extractions were carried out using distilled water and Na-phosphate buffer pH 7.0 as solvents and freeze\thaw and ultrasonication techniques were applied to optimize the extraction process. Extraction using the freeze\thaw method proved to be the most efficient method. Phycocyanin purification from crude extracts was carried out by a combination of methods such as ammonium sulfate (NH4)2SO4 65% precipitation, dialysis filter, and Sephadex G-100 gel filtration chromatography, to give 121.63 ± 0.03 mg\g C-PC content and 3.52 ± 0.04 purity. Antioxidant activity in vitro was determined by nitric oxide (NO) scavenging activity, percentage inhibition of NO by the C-PC in concentration 10 μg\mL to 100 μg\mL which significantly scavenged 55.89% to 91.54% of NO radicals, respectively. They exhibited strong NO radical scavenging capacity, the C-PC and vitamin C were able to reduce the stable NO radical to 50% reduction with EC50 of 4.53 and 3.66 µg\mL. Research results showed that S. platenis dry biomass provided by Dalitra was potential for obtaining C-phycocyanin (12.16% of dry biomass) and it was evaluated as an antioxidant in vitro able to scavenge nitric oxide. C-PC (12.16% of dry biomass) and was evaluated as an antioxidant in vitro able to scavenge nitric oxide.

Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles.

Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at -80°C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40°C in lyophilizates containing 10mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented.

PREPARATION OF S-ENANTHIOMERS OF 3-HYDROXY-1,4-BENZDIAZEPIN-2-ONE ESTERS BY BIOTECHNOLOGICAL WAY

Aim. Improving the method of obtaining the S-enantiomer of 3-acetoxy-5-phenyl7-bromo-1,2-dihydro-3H-1,4-benzdiazepin-2-one using microsomal fraction carboxylesterase included in agar. Methods. The microsomal fraction was obtained by low-speed sedimentation in the presence of calcium ions, the protein content was determined by the Lowry-Hartree method and esterase activity - according to 1-naphthyl acetate. Obtaining a reusable biocatalyst was performed by including the microsomal fraction in agar gel. Enzymatic hydrolysis of 3-acetoxy-5-phenyl-7-bromo-1,2-dihydro-3H-1,4-benzdiazepin-2-one was performed for 2.5 h in a solution of methyl cellosolve: Na-phosphate buffer, 0.0167 M, pH 7.0 (2/3, vol./ vol.), at a temperature of 37 ºC. Enantiomeric excess was determined by HPLC. Results. The method of obtaining the S-enantiomer of 3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H-1,4-benzdiazepin-2-one was improved. The inclusion of the microsomal fraction in the agar gel contributed to the development of an available method for obtaining a biocatalyst of multiple actions with high esterase activity (90%). The choice of methylcelosolve as a solvent in the enantioselective hydrolysis reaction made it possible to eliminate four stages of biotechnology for obtaining the S-enantiomer of the substrate. A modification of the carrier for column chromatography was performed. It allowed to visualize the process of separation of 1,4-benzdiazepin-2-one derivatives during chromatography using UV-irradiation. Chromatography system was changed from chloroform: 1,4-dioxane: heptane (2: 0.5: 2) to chloroform: acetonitrile (3: 0.5). This resulted in significantly better separation of 1,4-benzdiazepin-2-one derivatives. Conclusion. The method of obtaining the S-enantiomer of 3-acetoxy-7-bromo-5-phenyl-1,2-dihydro-3H1,4-benzdiazepin-2-one has been improved, which made it possible to simplify the process of its isolation by 5 stages.

Sequential recovery of C-phycocyanin and chlorophylls from Anabaena cylindrica

Cyanobacteria are attracting the attention worldwide as a reliable and sustainable feedstock for the production of biofuels, food colorants and biochemically active compounds. Anabaena cylindrica (A. cylindrica) is a cyanobacterium with a significant widespread occurrence in Portuguese freshwaters, with C-phycocyanin and chlorophylls, compounds with a high market value, being abundantly produced by the species. This work presents the development of a sequential downstream process to obtain C-phycocyanin and chlorophyll from A. cylindrica. The C-phycocyanin extraction is carried using Na-phosphate (20 mM, pH 7) buffer, while aqueous solutions of surface-active compounds (250 mM), as well as ethanol, were screened for the extraction of the chlorophylls. After selecting the best solvents to recover both pigments from A. cylindrica, the operational conditions (solid-liquid ratio, time of extraction and temperature) were optimized, allowing the recovery of around 90% and 55% of the total content of C-phycocyanin and chlorophylls, respectively. Finally, dextran/copolymer-based ABS were applied to promote the purification of C-phycocyanin from the residual chlorophylls and other contaminant proteins, leading to an increase in the C-phycocyanin purity of 4-fold, without compromising its photostability.

Enzyme protection systems of erythrocytes in conditions of ascorbate recirculation and oxidative loading

Vitamin C was shown to partially protect red blood cells from oxidative changes during storage by noticeable reduction of mechanical fragility and hemolysis. In order to maintain the content of ascorbate in the reconstituted form in plasma, the latter is involved in a number of oxidative-reducing processes within red blood cells. This work is a continuation of studies of the effects of ascorbate on the metabolic processes that maintain the viability of red blood cells. Human red blood cells were incubated for five hours at 25 ºC in the oxidizing media system 1 1.0 · 10–4 M ascorbic acid (AscH), 5 · 10–6 M Cu2+, Na-phosphate buffer (0.015 M, pH 7.4), 0.15 M NaCl, and system 2, that contained o-phenanthroline at a concentration of 1.0 · 10–4 M in addition to the components of system 1 medium. For these cells, the changes in the content of reduced glutathione, glutathione enzyme activity, and the state of the membrane electron transport NADH: ferricyanide reductase were determined in time. The obtained data indicate that red blood cells undergo significant oxidative stress under the influence of the oxidative medium. During the first incubation period of erythrocytes in the AscH-Cu2+ environment, the activity of glutathione peroxidase and glutathione-S-transferase reached the maximum values, indicating the presence of H2O2 in the cell and the activation of lipid peroxidation processes. Glutathione-S-transferase activity remained above the control level throughout the entire study period. The activity of glutathione reductase and glucose-6-phosphate dehydrogenase was reduced. The oxidative loading of erythrocytes in the presence of o-phenanthroline was lower, the development of oxidative stress occurred in 90 minutes, but the binding of the o-phenanthroline complexes of Cu2+ to the membrane modified the SH-group of membrane proteins and this reduced the transport capabilities of the dehydroascorbate transporters and the electron transmembrane system, the consequence of which may be the accumulation of oxidized forms of ascorbate outside. We detected the participation of CO-signaling mechanism in hemoglobin deglutathionylation and increase in the content of glutathione. In this work we discuss the role of metabolic reprogramming in red blood cells through thiol-disulfide exchange as a mechanism that can be involved into adaptive responses aimed at counteracting stress in mammalian tissues.

CARBOXYLESTERASES OF RAPANA VENOSA DIGESTIVE GLANDS HOMOGENATES

Aim. Investigation of biochemical and physico-chemical properties of carboxylesterases in homogenates gland and hepatopancreas of Rapana venosa; study of the features of enantioselective enzymatic hydrolysis of 3-hydroxy-1,4- benzodiasepine-2-one ester, potential anxiolytic and hypnotic drug. Methods. Digestive glands of Rapana venosa were homogenized for subsequent investigations, the protein content in them was determined by the Lowry-Hartree method and esterase activity – according to 1-, 2-naphthyl acetates. Enzymatic hydrolysis of 3-hydroxy-5-phenyl-7-bromo-1,2-dihydro-3H-1,4-benzodiasepine-2-one was conducted for 2,5 h in the solution: dimethyl sulfoxide: Na-phosphate buffer 0,0167 M, pH 7,0 in volume ratio 2:3, at 37 °C. Determination of enantiomeric excess was conducted with help of high performance liquid chromatography in SHIMATSU system, equipped with ChіraDex HR 5μm(4mm×250mm) column. Results. For the first time it was shown, that esterase activity of hepatopancreas and esophageal gland homogenates for 2-naphtyl acetate as substrate, was 3,6-fold and 6,7- fold greater, than for 1-naphtyl acetate, respectively. The belonging of esterases in homogenates to the family of carboxylesterase was confirmed by the total suppression of their activity by the selective carboxylesterase inhibitor, di-(p-nitrophenyl)phosphate (2.0 mmol/dm3). It was shown, that pH-optima of esterase activity in homogenates of esophageal gland and hepatopancreas equals 7,5 and 5,5, respectively. The features of 3-hydroxy-1,4-benzodiazepine-2-one ester hydrolysis, catalyzed by carboxylesterase of Rapana venosa digestive glands homogenates were studied. The preferential formation of the R-enantiomer of substrate was shown (enantiomeric excess of R- enantiomer was 42%). Conclusion. The regioselectivity of carboxylesterase to 1-, 2-naphtyl acetates and enantioselectivity of 3-hydroxy-1,4-benzodiazepine-2-one ester were found.

A change in the pathway of dithiothreitol-induced aggregation of bovine serum albumin in the presence of polyamines and arginine

When studying the anti-aggregation activity of chemical chaperones, a kinetic regime of the aggregation process for selected test systems should be established. To elucidate the mechanism of suppression of protein aggregation by polyamines (putrescine, spermidine) and arginine, we used a test system based on dithiothreitol (DTT)-induced aggregation of bovine serum albumin (BSA) at 45°C (0.1M Na-phosphate buffer, pH 7.0; [DTT]=2mM). The rate-limiting stage of DTT-induced aggregation of BSA under the studied conditions is that of unfolding of the protein molecule. The kinetics of BSA aggregation was monitored by dynamic light scattering and asymmetric flow field-flow fractionation. On the basis of the obtained data a mechanism of DTT-induced aggregation of BSA in the presence of polyamines and arginine has been proposed. It is assumed that the chemical chaperones under study stabilize the native form of protein with a subsequent decrease in the aggregation rate. However, they stimulate the sticking of aggregates formed in the aggregation process. To prove this mechanism, plots of the hydrodynamic radius of protein aggregates versus the portion of aggregated protein have been constructed.

Fabrication of Porcine DNA Biosensor Based on Ruthenium Bipyridine Complex

Electrochemical DNA biosensor for detection of porcine oligonucleotides based on ruthenium (II) as label redox complex has been developed. The system of biosensor is based on the modified screen printed carbon electrode (SPE) with goldnanoparticles (AuNPs) as transducer immobilized with poly(n-butylacrylate-N-acryloxysuccinimide) microsphere and porcine DNA probe sequences was attached onto it via the covalent bond. The ruthenium(II) complex, [Ru(bpy)2PIP]2+ PIP = 2-phenylimidazo[4,5-f[1,10-phenanthroline] intercalator has been been used to determine prcine DNA. The biosensor was measured and optimized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The interaction of [Ru(bpy)2PIP]2+ with various DNA with different sequences, i) single stranded probe DNA, ii) after hybridization with its complementary DNA and iii) after hybridization with mismatch complementary DNA were studied. The results indicated that the interaction of [Ru(bpy)2PIP]2+ with hybridized complementary DNA gave the highest response. Thus, development of porcine DNA biosensor and influences of many factors such as [Ru(bpy)2PIP]2+ complex and DNA probe concentration, DNA probe immobilization and hybridization time, pH, ionic strength, buffer concentration and temperatures were also studied to evaluate the performance of biosensor. The concentration of [Ru(bpy)2PIP]2+ complex and DNA probe were found to be optimal at 50 µM and 2 µM, respectively. The optimal time for DNA probe immobilization and hybridization were 7 hours and 60 minutes, accordingly. The optimal condition of pH, ionic strength, buffer concentration and temperatures were at pH 7.0, 1.0 M NaCl, 0.05 M of Na-phosphate buffer and 25 °C, respectively. The linear range of different concentration complementary DNA was within the range between 1.0 x 10-13 M to 1.0 x 10-8 M. This study was first reported the used of [Ru(bpy)2PIP]2+ for detection of porcine oligonucleotides.

Proteinases from the Liver of Albacore Tuna (Thunnus Alalunga): Optimum Extractant and Biochemical Characteristics

Proteolytic activity of the extract from albacore tuna (Thunnus alalunga) liver was studied. Optimum pH and temperature for casein hydrolysis were 8.5 and 55C, respectively. The enzyme was stable to heat treatment up to 50C and in the pH range of 7.0–10.0 for 30–120 min. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride. Activities of the liver extract continuously decreased with increasing NaCl concentration (0–30%), while activities increased as CaCl2 concentration increased. Based on activity staining, the molecular weights of the proteinases in albacore tuna liver were 21, 24, 30 and 34 kDa. Optimum extraction medium for proteinase recovery from albacore tuna liver was also investigated. Extraction of the liver powder with 50 mM Na phosphate buffer (pH 7.0) containing 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in albacore tuna liver were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. Practical Applications High amount of albacore tuna liver–containing proteinases is generated during canned tuna manufacturing. Major proteinases found in albacore tuna liver were trypsin-like serine proteinase that can be recovered and used mainly for hydrolysis purposes. Therefore, it is expected that albacore tuna liver can be used as a promising source of proteinase for further applications.

Kinetic regime of dithiothreitol-induced aggregation of bovine serum albumin

A search for agents, which are capable of effectively suppressing protein aggregation, and elaboration of the appropriate test systems, are among important problems of modern biochemistry and biotechnology. One such test system is based on dithiothreitol (DTT)-induced aggregation of bovine serum albumin (BSA). Study of the kinetics of DTT-induced aggregation of BSA by asymmetric flow field flow fractionation showed that a decrease in the portion of the non-aggregated protein in time followed the exponential law, the rate constant of the first order remaining unchanged at varying protein concentration (0.1M Na-phosphate buffer, pH 7.0; 45°C). The obtained results indicate that the rate-limiting stage of the general aggregation process is that of unfolding of the protein molecule. When studying the kinetics of DTT-induced aggregation of BSA by dynamic light scattering, we proposed to use parameter KLS as a measure of the initial rate of aggregation. Parameter KLS corresponds to the initial slope of the dependence of (I−I0)0.5 on time (I0 and I are the initial and current values of the light scattering intensity, respectively). The KLS value has been applied to estimate anti-aggregation activity of chemical chaperones (arginine, its derivatives and proline).

Вплив біоактивних альдегідів на властивості желатини

Bioactive aldehydes are among main factors of proteins postsynthetic modifications, which are the cause and consequence of many diseases. Comparative study of some aldehydes modifying action on gelatin was carried out in vitro. Gelatin samples (20 mM) were incubated with: ribose, deoxyribose, glyoxal, methylglyoxal, formaldehyde, acrolein (20 mM each) and their combinations in 0.1 M Naphosphate buffer (pH 7.4) containing 0.02% sodium azide at 37 °C in the dark for 30 days. We investigated the fluorescent properties of these samples and their molecular weight distribution by electrophoresis. It has been revealed that formed adducts had different fluorescence spectra. According to fluorescence intensity these aldehydes were put in order: formaldehyde < methylglyoxal < acrolein <ribose < deoxyribose < glyoxal. The electrophoresis results showed fragments of gelatin molecular weight redistribution. By this index, the aldehydes rating was as follows: ribose < deoxyribose < acrolein < glyoxal < formaldehyde < methylglyoxal. Comparison of these two ratings indicates that aldehydes with a lower ability to form fluorescent adducts have higher ability to form intermolecular crosslinks. Therefore, the traditional clinical fluorescent test of a patients' skin surface for collagen crosslinks determination has to be verified by other tests for proteins postsynthetic modifications.

Interaction of ceruloplasmin with eosinophil peroxidase as compared to its interplay with myeloperoxidase: Reciprocal effect on enzymatic properties

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are involved in the development of halogenative stress during inflammation. We previously described a complex between MPO and ceruloplasmin (CP). Considering the high structural homology between MPO and EPO, we studied the latter's interaction with CP and checked whether EPO becomes inhibited in a complex with CP. Disc-electrophoresis and gel filtration showed that CP and EPO form a complex with the stoichiometry 1:1. Affinity chromatography of EPO on CP-agarose (150 mM NaCl, 10 mM Na-phosphate buffer, of pH 7.4) resulted in retention of EPO. EPO protects ceruloplasmin from limited proteolysis by plasmin. Only intact CP shifted the Soret band typical of EPO from 413 to 408 nm. The contact with CP likely causes changes in the heme pocket of EPO. Peroxidase activity of EPO with substrates such as guaiacol, orcinol, o-dianisidine, 4-chloro-1-naphtol, 3,3’,5,5’-tetramethylbenzidine, and 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) is inhibited by CP in a dose-dependent manner. Similar to the interaction with MPO, the larger a substrate molecule, the stronger the inhibitory effect of CP upon EPO. The limited proteolysis of CP abrogates its capacity to inhibit the peroxidase activity of EPO. The peptide RPYLKVFNPR (corresponding to amino acids 883–892 in CP) inhibits the peroxidase and chlorinating activity of EPO. Only the chlorinating activity of EPO is efficiently inhibited by CP, while the capacity of EPO to oxidize bromide and thiocyanate practically does not depend on the presence of CP. EPO enhances the p-phenylenediamine-oxidase activity of CP. The structural homology between the sites in the MPO and EPO molecules enabling them to contact CP is discussed.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Relationship between the initial rate of protein aggregation and the lag period for amorphous aggregation

Lag period is an inherent characteristic of the kinetic curves registered for protein aggregation. The appearance of a lag period is connected with the nucleation stage and the stages of the formation of folding or unfolding intermediates prone to aggregation (for example, the stage of protein unfolding under stress conditions). Discovering the kinetic regularities essential for elucidation of the protein aggregation mechanism comprises deducing the relationship between the lag period and aggregation rate. Fändrich proposed the following equation connecting the duration of the lag phase (tlag) and the aggregate growth rate (kg) in the amyloid fibrillation: kg=const/tlag. To establish the relationship between the initial rate of protein aggregation (v) and the lag period (t0) in the case of amorphous aggregation, the kinetics of dithithreitol-induced aggregation of holo-α-lactalbumin from bovine milk was studied (0.1M Na-phosphate buffer, pH 6.8; 37°C). The order of aggregation with respect to protein (n) was calculated from the dependence of the initial rate of protein aggregation on the α-lactalbumin concentration (n=5.3). The following equation connecting v and t0 has been proposed: v1/n=const/(t0−t0,lim), where t0,lim is the limiting value of t0 at high concentrations of the protein.

The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel

Crystal structures of the CorA Mg(2+) channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open "metal free" state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg(2+). We find that when Mg(2+) is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg(2+) is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83U/mg specific activity. Silver staining after SDS–PAGE revealed the presence a single band of 45kDa. The enzyme exhibited a temperature optimum of 60°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn2+, Ca2+, Mg2+, Cu2+, Mn2+) and stimulated by 50mM Fe2+. Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.