Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography

Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.

     

Selective capillary coating materials for in-tube solid-phase microextraction coupled to liquid chromatography to determine drugs and biomarkers in biological samples: A review

In-tube solid-phase microextraction (in-tube SPME) coupled with high performance liquid chromatography (HPLC) or liquid chromatography coupled to mass spectrometry (LC-MS) successfully determines drugs or biomarkers in biological samples by direct sample injection or by simple sample treatment. This technique uses a capillary column as extraction device. Several capillaries (wall-coated open tubular, sorbent-packed, porous monolithic rods, or fiber-packed) with unique phases have been developed and evaluated, aiming to improve the efficiency and selectivity of the in-tube SPME-LC technique. This review describes new developments and applications occurred in recent years, and discusses future trends with emphasis on new extraction devices and current technology used for the synthesis of selective sorbents for bioanalysis, such as (i) polypyrrole, (ii) restricted-access materials, (iii) immunosorbents, (iv) molecular imprinting polymers, (v) monolithic polymers, and (vi) bi-functional materials.     

Synthesis of diarylamines in the benzo[b]thiophene series bearing electron donating or withdrawing groups by Buchwald-Hartwig C-N coupling

Diarylamines in the benzo[b]thiophene series bearing electron donating or withdrawing groups, were prepared by Buchwald-Hartwig C-N coupling in moderate to high yields. The conditions used were Pd(OAc)₂ (3 mol%), BINAP as ligand (4 mol%) and Cs₂CO₃ as base (1.4 equiv.), in toluene at 100°C, being 6-bromo or amino benzo[b]thiophenes coupled, respectively, with substituted anilines or phenylbromides. The 6-aminobenzo[b]thiophene derivatives were also prepared by palladium catalyzed C-N coupling of the corresponding 6-bromo compounds with benzophenone imine, followed by acidic hydrolysis of the imino derivatives. When 4-nitrobromobenzene and 4-bromobenzonitrile were used as coupling components, triarylamines were also isolated in small amounts. The presence of a fluorine atom on the phenylbromide highly increases the diarylamine yields.     

Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA

Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.     

Effect of salts and temperature on the adsorption of bovine serum albumin on polypropylene glycol-Sepharose under linear and overloaded chromatographic conditions

The interaction thermodynamics associated with bovine serum albumin (BSA) adsorption on polypropylene glycol (PPG)-Sepharose CL-6B gel, using ammonium and sodium sulfate was studied. Analysis of data under linear conditions was accomplished with the stoichiometric displacement retention model and preferential interaction approach. Preferential interaction analysis indicated a strong entropic driving force due to the release of a large amount of solvent on adsorption. Flow microcalorimetry provided direct heat of adsorption measurements under overloaded conditions and confirmed that the adsorption of BSA on PPG-Sepharose was entropically driven within the range of conditions studied. Using these data in combination with isotherm measurements, it is shown that protein surface coverage, salt concentration, salt type and temperature affect the enthalpic and entropic behavior in hydrophobic interaction chromatography (HIC). This study shows that protein-sorbent interactions can be strongly influenced by the degree of water release, protein-protein interactions on the surface, and the re-orientation and/or reconfiguration of the adsorbed protein.     

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5‐HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC‐ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5‐HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R²) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5‐HT, and 5 and 2 ng/mL for DA and NE. Within‐ and between‐run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50–8.36 and 0.54–13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5‐HT, DA and NE in urine by coulometric detection. Copyright © 2012 John Wiley & Sons, Ltd.     

On dividing chains in simple theories

Dividing chains have been used as conditions to isolate adequate subclasses of simple theories. In the first part of this paper we present an introduction to the area. We give an overview on fundamental notions and present proofs of some of the basic and well-known facts related to dividing chains in simple theories. In the second part we discuss various characterizations of the subclass of low theories. Our main theorem generalizes and slightly extends a well-known fact about the connection between dividing chains and Morley sequences (in our case: independent sequences). Moreover, we are able to give a proof that is shorter than the original one. This result motivates us to introduce a special property of formulas concerning independent dividing chains: For any dividing chain there exists an independent dividing chain of the same length. We study this property in the context of low, short and ω -categorical simple theories, outline some examples and define subclasses of low and short theories, which imply this property. The results give rise to further studies of the relationships between some subclasses of simple theories. Research supported by CNPq grant 150309/2003-1. Research supported by CNPq grant 304365/2003-3 (Modelos, Provas e Algoritmos)     

On-line characterisation of apple polyphenols by liquid chromatography coupled with mass spectrometry and ultraviolet absorbance detection

Apple polyphenols were characterised by means of hyphenated techniques such as HPLC coupled to UV photodiode array detection (LC-DAD) and to mass spectrometry (LC-MS). LC-MS using atmospheric pressure ionisation (APCI) in the positive ion mode provided the molecular weight, the number of hydroxyl groups, the number of sugars and an idea about the substitution pattern of the polyphenols. LC-DAD with postcolumn addition of UV shift reagents afforded precise structural information about the position of the free hydroxyl groups in the polyphenolic nucleus. Five isorhamnetin glycosides, two hydroxyphloretin glycosides and quercetin were reported in apple peel for the first time. Postcolumn addition of UV shift reagents in LC-DAD analysis confirmed the presence of isorhamnetin glycosides and not the isomeric glycosides of rhamnetin. Moreover, isorhamnetin-3-O-rhamnoglucoside was identified unambiguously by comparison with a standard. These results are relevant not only from a chemotaxonomic point of view, but also in the control of authenticity of fruit derived products in order to detect fraudulent admixtures.     

Overproduction of Polygalacturonase by Penicillium griseoroseum Recombinant Strains and Functional Analysis by Targeted Disruption of the pgg2 Gene

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.     

Ethylenediamine-Derived Chromatographic Ligand to Separate BSA,Lysozyme, and RNase A

Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.