Molecular Exclusion Chromatography Research Articles

THU0011 Three Different Protein Products are Codified by Human MMP-13 Gene

BackgroundCollagenase-3 (MMP-13) is a human matrix metalloproteinase highly overexpressed in diseases where tissue repair and remodelling is needed, such as rheumatoid arthritis (RA), osteoarthritis (OA) and some cancers. However, its...

Verification of three different MMP-13 isoforms expression in human osteoarthritic cartilage

Verification of three different MMP-13 isoforms expression in human osteoarthritic cartilage

Ex vivo and in vivo magnetic resonance imaging of cartilage canals in swine at predilection sites of osteochondrosis

Ex vivo and in vivo magnetic resonance imaging of cartilage canals in swine at predilection sites of osteochondrosis

Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate.

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

In vitro anti-bacterial and anti-fungal activities of hydrophilic plant defence compounds obtained from the leaves of bell pepper (Capsicum annuum L.)

SummaryHydrophilic extracts from plants contain anti-microbial macromolecules that may be considered when developing natural compounds to control plant pathogens, including anti-microbial peptides that differ structurally from commercial pesticides. This study evaluated the anti-microbial activities of aqueous peptide-enriched fractions from the leaves of bell pepper (Capsicum annuum L. ‘Magali R’) against phytopathogens for biotechnological applications. Fully-expanded bell pepper leaves were frozen, powdered and extracted. The supernatant was labelled the soluble extract (SE), and the precipitate was extracted with LiCl to produce a cell wall extract (CWE). The SE and CWE were then fractionated using ammonium sulphate following molecular exclusion chromatography. Electrophoresis showed that the CWE was a less complex extract. A peptide-enriched fraction (PF) from the CWE was then fractionated by hydrophobic chromatography. Two of the fractions recovered inhibited the growth of Ralstonia solanacearum and Clavibacter michiganensis ssp. michiganensis in vitro. For commercial-scale applications, ultrafiltration (1.0 – 10.0 kDa cut-off) replaced chromatography, for lower cost. The peptide sub-fraction CWE1-10kDa partially controlled the in vitro growth of R. solanacearum, C. michiganensis ssp. michiganensis, and Alternaria solani and showed high activity against Erwinia carotovora ssp. carotovora, with total inhibition at a low concentration of 96.7 mg peptide fruction l–1. CWE1-10kDa represents an aqueous anti-microbial extract that may complement the use of pesticides, and have lower environmental toxicity.

Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase fromBothrops moojeniSnake Venom

A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh−1nmol−1 ± 0.1031 and K m = 14.59 mg/mL ± 4.610).

Purification, Characterization and Application of Polygalacturonase from Aspergillus niger CSTRF

Aims: The research was carried out to study the purification, characterization and application of polygalacturonase from Aspergillus niger CSTRF. Methodology and Results: The polygalacturonase (PG) from the fungus was purified by ammonium sulphate precipitation and dialysed. The resulting fraction of the enzyme was further separated by molecular exclusion and ion exchange chromatography. The enzyme was purified 28.19 fold with a yield of approximately 69 % following purification with SP C�50. It has a relative molecular weight of 79,430 daltons and markedly influenced by temperature, pH and substrate concentrations of reactions with optimum activity at 35 °C, pH 4.0 and 8 mg/mL respectively. The PG was heat +

LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

A basic phospholipase A2 (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery® Bio Wide column. From liquid chromatography–electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA2 LmrTX from L. muta rhombeata and other PLA2 from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA2 activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA2 from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin.

Substituted thiobenzoic acid S-benzyl esters as potential inhibitors of a snake venom phospholipase A2: Synthesis, spectroscopic and computational studies

4-Chlorothiobenzoic acid S-benzyl ester (I), 3-nitrothiobenzoic acid S-benzyl ester (II), 4-nitrothiobenzoic acid S-benzyl ester (III) and 4-methylthiobenzoic acid S-benzyl ester (IV) were prepared and characterized by 1H and 13C NMR, Mass spectrometry and IR spectroscopy. Quantum chemical calculations were performed with Gaussian 09 to calculate the geometric parameters and vibrational spectra. Phospholipase A2 (PLA2) was purified from Crotalus durissus cumanensis venom by molecular exclusion chromatography, followed by reverse phase-high performance liquid chromatography. Two studies of the inhibition of phospholipase A2 activity were performed using phosphatidilcholine and 4-nitro-3-octanoyloxybenzoic acid as substrates, in both cases compound II showed the best inhibitory ability, with 74.89% and 69.91% of inhibition, respectively. Average percentage of inhibition was 52.49%. Molecular docking was carried out with Autodock Vina using as ligands the minimized structures of compounds (I–IV) and as protein PLA2 (PDB code 2QOG). The results suggest that compounds I–IV could interact with His48 at the active site of PLA2. In addition, all compounds showed Van der Waals interactions with residues from hydrophobic channel of the enzyme. This interaction would impede normal catalysis cycle of the PLA2.

Purification and characterization of a bacteriocin from Lactobacillus rhamnosus L34

Bacteriocins are ribosomally synthesized peptides having considerable potential as a food preservative because of their strong antagonistic activity against many food spoilage and pathogenic organisms. A bacteriocin from Lactobacillus rhamnosus isolates was purified using ammonium sulphate precipitation and molecular exclusion chromatography techniques. Ammonium sulphate precipitation resulted in higher yield of bacteriocin, but the specific activity and fold purification were higher for molecular exclusion chromatography. The bacteriocin exhibited inhibition against food-borne pathogens and spoilage microorganisms, including both Gram-positive and -negative bacteria. Amylase, lipase and catalase did not alter the antimicrobial activity but proteolytic enzymes inactivated the bacteriocin. It was heat stable and exhibited activity in a pH range of 2–8 with maximum activity at pH 5.0. Molecular weight of bacteriocin was found to be ~5.6 kDa using SDS-PAGE. HPLC profile showed a single peak further attesting the purity of the bacteriocin.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Purification and Characterization of Peroxidase from Papaya (Carica papaya) Fruit

Ripening of papaya fruit was found to be characterized with a decrease in peroxidase activity and its transcript. This peroxidase was purified to homogeneity through successive steps of ammonium sulfate fractionation, ion exchange and molecular exclusion chromatography. The peroxidase was purified 30.22-folds with overall recovery of 44.37% and specific activity of 68.59. Purified peroxidase was found to be a heterotrimer of ~240 kDa, containing two subunits each of 85 and one of 70 kDa. Purified enzyme exhibited pH and temperature optima of 7.0 and 40 °C, respectively. K(m) values for substrates o-dianicidin, guaiacol and ascorbic acid were found to be 0.125, 0.8 and 5.2 mM, respectively. K(m) for H(2)O(2) was found to be 0.25 mM. Salicylic acid was found to activate peroxidase up to 50 μM concentration, beyond which it acted as inhibitor. Ca(2+) and Mg(2+) activated peroxidase while sodium azide, SDS, and Triton X-100 were found to inhibit peroxidase.

Separation of oligosaccharides from caprine milk whey, prior to prebiotic evaluation

Milk oligosaccharides are believed to have beneficial biological properties. Caprine milk has a relatively high concentration of oligosaccharides in comparison with other ruminant milks and has the oligosaccharide profile closest to that of human milk. The first stage in recovering oligosaccharides from caprine milk whey, a by-product of cheese making, was accomplished by ultrafiltration to remove proteins and fat globules, leaving more than 97% of the initial carbohydrates, mainly lactose, in the permeate. The ultrafiltered permeate was further processed using a 1 kDa ‘tight’ ultrafiltration membrane, which retained less than 7% of the remaining lactose. The final retentate was fractionated by preparative scale molecular size exclusion chromatography to yield 28 fractions; oligosaccharide-rich fractions that were suitable for functionality and gut health promotion testing were detected between fractions 9/10 to 16/17. All fractions were evaluated for their oligosaccharide and carbohydrate profiles using three complementary analytical methods.

Ficus sycomorus latex: A thermostable peroxidase

Peroxidase from sycamore fig Ficus sycomorus latex (POLI) was purified by heat treatment, anion exchange chromatography and molecular exclusion chromatography. The purity was determined from high specific activity (9166 units/mg protein), purification fold (28), RZ value 3.1 and a single band in native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and visualized peroxidase activity on the PAGE. POLI had molecular mass of 43 kDa. Substrates commonly used in immunodiagnostic kits as 2,2-azino-bis [3-ethyl- benzothiazoline-(6)-sulfonic acid] (ABTS), 4-chloro-1- naphthol (4C-1N), o-phenylenediamine (OPD) and 3,3`,5,5`- tetramethylbenzidine (TMB) were found to be the best substrates for the enzyme. The K m for catalysis of H 2 O 2 was 1.2 mM. The catalytic efficiency (Vmax/Km) for POLI was found to follow the order: ABTS, 4C-1N, OPD, TMB, guaiacol, paminoantipyrine, o-dianisidine and pyrogallol. The enzyme showed a broad pH optimum ranged from pH 5.5 to 7.0. The optimal temperature for the enzyme was 35 to 40°C. POLI showed highest thermal stability. No loss of enzyme activity was recorded up to 60°C, whereas only 20% of enzyme activity was lost at 70 to 90°C. The thermal inactivation profiles of POLI demonstrated that the enzyme had higher thermal resistance. The peroxidase activity was slightly enhanced by low concentration of Ca 2+ , Ni +2 and Mg 2+ and high concentration of Mn 2+ . Fe +3 , Zn +2 , Hg +2 caused slightly inhibitory effects. In conclusion, sycamore fig latex will be a new and potential source for a peroxidase enzyme. Key words: Ficus sycomorus, latex, peroxidase, purification, characterization.

Purification and inflammatory edema induced by two PLA2 (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA(2) were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on μ-Bondapak C18 column. Both PLA(2) showed a molecular mass of ~14kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA(2)s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA(2) classification scheme to arise. In addition, Anch TX-I and II, despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA(2) (Anch TX-I and III) showed high amino acid sequence identity with PLA(2)Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA(2) of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA(2) from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PLA(2). We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema.

Structural and Functional Characterization of a &amp;#947;-Type Phospholipase A2 Inhibitor from Bothrops jararacussu Snake Plasma

Phospholipases A2 (PLA2s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between γBjussuMIP (MIP) and Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP.

Purification and properties of an acid β-xylosidase from Penicillium sclerotiorum

The β-xylosidase from Penicillium sclerotiorum was purified to homogeneity by a rapid and inexpensive procedure involving ammonium sulphate fractionation and molecular exclusion chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed two bands with an estimated molecular mass of 97 and 42 kDa, respectively. Electrophoretical homogeneity was observed under non-denaturing PAGE conditions. These results indicate that this protein has a dimeric structure. The molecular mass of the native enzyme estimated by molecular exclusion chromatography was 144 kDa. The enzyme is a glycoprotein with a 56.4% carbohydrate content. The pH and temperature optima were 2.5 and 60°C, respectively. The enzyme remained stable over a pH range from 2.0 to 7.0 and at temperatures up to 60°C for 375 min. All divalent cations tested, except for Hg2+, inhibited β-xylosidase activity, especially at a concentration of10 mM. The purified enzyme was also sensitive to denaturing agents SDS and EDTA and was activated by thiol-containing reducing agents. The Michaelis–Menten constant for p-nitrophenyl-β-D-xylopyranoside was 0.78 mM, and the maximum reaction velocity was 0.51 μmole of p-nitrophenol min-1 mg-1 of protein. This is the first report on the purification and characterization of a β-xylosidase from P. sclerotiorum, which has potential applications in a number of biotechnological processes, such as animal feed, juice and wine industries.

Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A2s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD50s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD50 of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD50s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bβ chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.

Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus)

An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55°C. The enzyme was completely inactivated after 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km-1 values, using BApNA as substrate, were 0.266mM, 0.93s−1 and 3.48mM−1s−1, respectively. Enzyme activity increased in the presence of the ions (1mM) K+, Li+ and Ca2+, but was inhibited by Fe2+, Cd2+, Cu2+, Al3+, Hg2+, Zn2+ and Pb2+ as well as by the trypsin inhibitors TLCK and benzamidine.

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom

BackgroundMultifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms. MethodsThe l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity. ResultsBl-LAAO is a 60kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H2O2. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC50 of 0.07μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H2O2 in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase. ConclusionBl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H2O2 which kill the cells. General significanceThese results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.