Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Immobilized Metal Ion Affinity Cromatography - IMAC - is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.

Extraction, purification and biochemical characterization of a peroxidase from Copaifera langsdorffii leaves

The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost.

CROMATOGRAFIA DE AFINIDADE COM CORANTE RED A versus TROCA IÔNICA-PERMEABILIDADE EM GEL: COMPARAÇÃO DA PRATICIDADE NA PURIFICAÇÃO DE ENTEROTOXINA ESTAFILOCÓCICA A

Culture supernatant of Staphylococcus aureus 722 in 3% triptone plus 1% yeast extract was used for EEA purification, proceeding comparison between dye ligand Red A affinity chromatography and classic chromatography. The capture of SEA with Amberlite CG-50 allowed rapid enterotoxin concentration from the culture supernatant. However, the ratio of 15 mg of the resin to a total of 150 mg of the toxin satured the resin, giving only 10 to 30% of SEA recuperation from the supernatant. The elution of concentrated material throught the Red A column resulted in a recovery of 60,87% of the toxin, and required 76 hours, indicating advantage on classic chromatography. Ion exchange column plus gel filtration recovered only 6,5 % of the SEA, and required 114 hours to conclude the procedure. The eletrophoresis of purified SEA indicated high grade of toxin obtained from Red A column, with 90 % of purity, compared to 60 % of classic column.

Assessment Of Robustness On Analysis Using Headspace Solid-phase Microextraction And Comprehensive Two-dimensional Gas Chromatography Through Experimental Designs.

Plackett-Burman experimental design was applied for the robustness assessment of GC×GC-qMS (Comprehensive Two-Dimensional Gas Chromatography with Fast Quadrupolar Mass Spectrometric Detection) in quantitative and qualitative analysis of volatiles compounds from chocolate samples isolated by headspace solid-phase microextraction (HS-SPME). The influence of small changes around the nominal level of six factors deemed as important on peak areas (carrier gas flow rate, modulation period, temperature of ionic source, MS photomultiplier power, injector temperature and interface temperature) and of four factors considered as potentially influential on spectral quality (minimum and maximum limits of the scanned mass ranges, ions source temperature and photomultiplier power). The analytes selected for the study were 2,3,5-trimethylpyrazine, 2-octanone, octanal, 2-pentyl-furan, 2,3,5,6-tetramethylpyrazine, and 2-nonanone e nonanal. The factors pointed out as important on the robustness of the system were photomultiplier power for quantitative analysis and lower limit of mass scanning range for qualitative analysis.

Propylthiouracil Quantification In Human Plasma By High-performance Liquid Chromatography Coupled With Electrospray Tandem Mass Spectrometry: Application In A Bioequivalence Study.

A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS) in negative mode (ES-). Chromatography was performed using a Phenomenex Gemini C18 5μm analytical column (4.6mm×150mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v)+0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 (V), -26 (eV) and -5 (V), respectively. The method had a chromatographic run time of 2.5min and a linear calibration curve over the range 20-5000ng/mL. The limit of quantification was 20ng/mL. The stability tests indicated no significant degradation. This HPLC-MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63-115.25%) and 115.60% (109.03-122.58%) for Cmax, 103.31% (100.74-105.96%) and 103.40% (101.03-105.84) for AUClast. This method offers advantages over those previously reported, in terms of both a simple liquid-liquid extraction without clean-up procedures, as well as a faster run time (2.5min). The LOQ of 20ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the Tmax and decreased the bioavailability (Cmax and AUC).

Bp-13 Pla2: Purification And Neuromuscular Activity Of A New Asp49 Toxin Isolated From Bothrops Pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca(2+) by Sr(2+) and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.

In Vivo Determination Of The Volatile Metabolites Of Saprotroph Fungi By Comprehensive Two-dimensional Gas Chromatography.

In this work, we discuss the use of multi-way principal component analysis combined with comprehensive two-dimensional gas chromatography to study the volatile metabolites of the saprophytic fungus Memnoniella sp. isolated in vivo by headspace solid-phase microextraction. This fungus has been identified as having the ability to induce plant resistance against pathogens, possibly through its volatile metabolites. Adequate culture media was inoculated, and its headspace was then sampled with a solid-phase microextraction fiber and chromatographed every 24 h over seven days. The raw chromatogram processing using multi-way principal component analysis allowed the determination of the inoculation period, during which the concentration of volatile metabolites was maximized, as well as the discrimination of the appropriate peaks from the complex culture media background. Several volatile metabolites not previously described in the literature on biocontrol fungi were observed, as well as sesquiterpenes and aliphatic alcohols. These results stress that, due to the complexity of multidimensional chromatographic data, multivariate tools might be mandatory even for apparently trivial tasks, such as the determination of the temporal profile of metabolite production and extinction. However, when compared with conventional gas chromatography, the complex data processing yields a considerable improvement in the information obtained from the samples. This article is protected by copyright. All rights reserved.

Profile Of Phenolic Compounds Of Brazilian Virgin Olive Oils By Rapid Resolution Liquid Chromatography Coupled To Electrospray Ionisation Time-of-flight Mass Spectrometry (rrlc-esi-tof-ms).

In recent years, agronomical researchers began to cultivate several olive varieties in different regions of Brazil to produce virgin olive oil (VOO). Because there has been no reported data regarding the phenolic profile of the first Brazilian VOO, the aim of this work was to determine phenolic contents of these samples using rapid-resolution liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry. 25 VOO samples from Arbequina, Koroneiki, Arbosana, Grappolo, Manzanilla, Coratina, Frantoio and MGS Mariense varieties from three different Brazilian states and two crops were analysed. It was possible to quantify 19 phenolic compounds belonging to different classes. The results indicated that Brazilian VOOs have high total phenolic content because the values were comparable with those from high-quality VOOs produced in other countries. VOOs from Coratina, Arbosana and Grappolo presented the highest total phenolic content. These data will be useful in the development and improvement of Brazilian VOO.

Preparative separation of flavonoids from the medicinal plant Davilla elliptica St. Hill. by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) is a major tool for the fast separation of natural products from plants. It was used for the preparative isolation of the flavonoid monoglucosides present in the aerial parts of the Davilla elliptica St. Hill. (Dilleniaceae). This species is used in Brazilian folk medicine for the treatment of gastric disorders. The optimum solvent system used was composed of a mixture of ethyl acetate-n-propanol-water (140:8:80, v/v/v) and led to a successful separation of quercetin-3-O-alpha-L-rhamnopyranoside and myricetin-3-O-alpha-L-rhamnopyranoside in approximately 3.0 hours with purity higher than 95%. Identification was performed by ¹H NMR, 13C NMR and HPLC-UV-DAD analyses.

The syntheses of the marine metabolites 3-bromoverongiaquinol and 5-monobromocavernicolin

An efficient synthesis of the marine metabolite 3-bromoverongiaquinol (1) and the first total synthesis of 5-monobromocavernicolin (2), both isolated from the marine sponge Aplysina cavernicola, have been described based on the 1,2 addition of the lithium enolate of N,O-bistrimethylsilylacetamide (BSA, 4) to 1,4-benzoquinone (3). Bromination and purification of the crude product on silica gel chromatography provided 3-bromoverongiaquinol (1) in 50% overall yield. Under alkaline conditions, the crude product of the bromination reaction was converted to 5-monobromocavernicolin (2) in 20% yield which was also obtained in 13% yield (25% yield based on recovered starting material) from 3-bromoverongiaquinol (1).