Name: preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation
Uploaded: 2018-03-09T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Esta pesquisa foi apoiada pelo programa de pesquisa de ciência básica (número do projecto: 2016R1A2B4013467) através do nacional pesquisa Fundação da Coreia (NRF) financiado pelo Ministério da ciência, TIC & futuro planejamento 2016 e também apoiada pela ciência básica de Coreia Instituir o programa de pesquisa (número do projecto: C36707).

1. síntese de DMMTAV
<ol><li>
Preparação química e razão molar mistura de DMAVat2S e H2SO4 Nota: DMAV: at2S:H2então4 = 1:1.6:1.6<ol>
<li>Dissolver 5,24 g de DMAV em 40 mL de água deionizada e N2-expurgados (descartados pelo menos 30 min) água num tubo de centrífuga de 50 mL.</li>
		<li>Preparar o reagente de2S at dissolvendo 14,41 g de at2S·9H2O em 50 mL de água deionizada e N2-expurgados de água em um balão de 250 mL.</li>
		<li>Preparar H2para4 reagente adicionando 3,3 mL de ácido sulfúrico concentrado (96%) para 40 mL de água deionizada e N2-expurgados de água em um tubo de centrífuga de 50 mL. Nota: A relação molar Final de DMAV: Na2S:H2SO4 = 1:1.6:1.6 1,3,4,8,15.</li>
		<li>Adicione o preparado 40 mL da solução DMAV (etapa 1.1.1) para os 50 mL de at2S solução contida no frasco de 250 mL (passo 1.1.2). Enxaguar o tubo contendo DMAV com 10 mL de água purgada e adicionar esta para o balão de 250 mL também.</li>
		<li>Feche o frasco com uma rolha de borracha do três-furo equipada com tubos de vidro. Use o vidro tubos de influxo de gás N2 , vazão e H2entrada de solução SO4 , respectivamente. Imediatamente depois de fechar o frasco, permita o fluxo de gás de2 N para o balão. Nota: A pressão do gás deve ser mantida a fluir sobre a superfície da solução de reação sem salpicos.</li>
		<li>Conectar-se resistente aos ácidos de tubos para o tubo de vidro do H2entrada de solução4 SO com uma seringa de 50 mL, adicionar o preparado de 40 mL de H2então4 solução (etapa 1.1.3). Adicionar 40 mL de H2então4 solução, lentamente e de forma gradual. Atenção: Após a adição de ácido sulfúrico, fumos brancos serão gerados; Use uma coifa bem ventilada.</li>
		<li>Monitorar a mudança de cor da mistura no recipiente de reação, enquanto a adição de ácido sulfúrico em intervalos regulares. Manter um intervalo de 4-5 mL gotas de H2então4; a mistura deve ser uma solução turva branca. Nota: Precipitação instantânea de amarela pode aparecer devido a rápida adição de ácido sulfúrico concentrado.</li>
		<li>Certifique-se de que solução a reação tem sido permanente por 1h, desde o início da etapa 1.1.4.</li>
	</ol>
</li>
	<li>
Extração deV DMMTA usando o método de extração líquido-líquido
	<ol>
<li>Após 1 h, despeje a solução de reação em um funil de separação contendo cerca de 200 mL de éter dietílico.</li>
		<li>Agite o funil durante 5-10 min, liberando gás várias vezes, alternando a torneira. Nota: Sintetizado DMMTAV vai ser transferido para a camada superior de éter dietílico (0.713 g·mL-1). Atenção: Gás de éter dietílico podem ser prejudicial; Use uma coifa bem ventilada.</li>
		<li>Recolher a solução de reação num copo e recolher separadamente o éter dietílico contendo DMMTAV em uma garrafa. Coloque a solução de reação volta no mesmo funil de separação e adicionar cerca de 200 mL de fresco éter dietílico para reshaking. Repita os passos 1.2.2 - 1.2.3 três vezes.</li>
		<li>Despeje o éter dietílico coletado da etapa 1.2.3 no funil de separação mesmo novamente e adicionar cerca de 100 mL N2-expurgados de água desionizada. Agitar durante 5-10 min e descartar o N2-expurgados de água desionizada e alguns mL de éter dietílico para fins de pureza. Colete o restante éter etílico em um prato de petri de vidro (diâmetro interno mínimo de 160 mm) e altura mínima de 50 mm.</li>
		<li>Transferi o prato de petri de vidro em uma caixa de luva N2 atmosfera para impedir a transformação de espécies. Atenção: Certifique-se que o solvente não é puxado para a bomba de vácuo através da saída da caixa de passagem.</li>
		<li>Seco até um precipitado branco de dimethylmonothioarsinate (Vde DMMTA cristalizada) é formada sobre o prato de petri de vidro. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Verificação de armazenamento e sintetizado DMMTAV
	<ol>
<li>Leve o precipitado branco de cristalizada DMMTAVe medir e registrar seu peso total. Atenção: Use uma coifa bem ventilada ou luvas para evitar a inalação de gás sulfeto de hidrogênio.</li>
		<li>Dissolver cristalizado DMMTAV em 50 mL de N2-purga de água desionizada e filtrar o precipitado amarelo através de um filtro de seringa 0,2 µm.</li>
		<li>Assumir que o total a partir DMA usadosV é convertido em DMMTAV, ou seja,≈9, 649 mg As· L-1. Diluir a solução-mãe DMMTAV para ≈1 mg· L-1 e ≈40 µg· L-1 para análise de verificação utilizando ionização Electrospray Mass Spectromtery (ESI-MS) e HPLC-ICP-MS, respectivamente.</li>
		<li>Analise a m/z de DMMTAV utilizando ESI-MS11,16 e fragmentos, em m/z 155 no modo positivo-íon ou em m/z 153 no modo negativo-íon (tabela 1). Nota: Ver referência os valores de m/z (tabela 1).</li>
		<li>Cromatograma do DMMTAV na solução estoque usando HPLC-ICP-MS11,16,17 com eluente adequadas condições de analisar e confirmar um grande pico encontra-se no tempo de retenção, descrito no literatura. Nota: Pureza de sintetizado DMMTAV deve ser calculada utilizando os resultados da análise da etapa 1.3.5.</li>
		<li>Analisar a concentração de arsênio total usando ICP-MS após digestão com ácido11 e calcular a concentração real de DMMTAV em solução-mãe de DMMTAV sintetizada usando fatores de diluição e pureza, como na seguinte equação: Total analisado como concentração (µg· L-1) · · Fator de diluição Pureza (%) = concentração de verdade DMMTAV DMMTAV solução estoque (µg· L-1)</li>
		<li>Armazenar a solução estoque de DMMTAV a 4 ° C no escuro por mais análise quantitativa especiação18.</li>
	</ol>
</li>
</ol>2. síntese de DMDTAV
<ol><li>
Preparação química e razão molar mistura de DMAV at2S e H2SO4 Nota: DMAV: at2S:H2então4 = 1:7.5:7.5<ol>
<li>Dissolva 1,38 g de DMAV em 40 mL de água desionizada num tubo de centrífuga de 50 mL.</li>
		<li>Prepare o reagente de2S at dissolvendo 18,01 g de Na2S·9H2O em um 50 mL de água desionizada num balão de 250 mL.</li>
		<li>Prepare-se H2para que4 reagente adicionando 4 mL de ácido sulfúrico concentrado (96%) para 40 mL de água desionizada contido num tubo de centrífuga de 50 mL. Nota: A relação molar Final de DMAV: Na2S:H2SO4 = 1:7.5:7.51,3,4,8,15.</li>
		<li>Adicione o preparado 40 mL da solução DMAV (etapa 2.1.1) para os 50 mL de at2S solução contida no frasco de 250 mL (passo 2.1.2). Enxaguar o tubo contendo DMAV com 10 mL de água desionizada e adicionar esta para o balão de 250 mL também.</li>
		<li>Adicionar o preparado 40 mL de H2então4 solução (etapa 2.1.3), lentamente e de forma gradual, para o balão.</li>
		<li>Monitore a mudança de cor da mistura no recipiente de reação, enquanto a adição de ácido sulfúrico em intervalos regulares. Manter um intervalo de 4-5 mL gotas de H2então4; a mistura deve ser uma solução turva branco/amarelo. Nota: Precipitação instantânea de amarela pode aparecer devido a uma rápida adição de ácido sulfúrico concentrado. Atenção: Após a adição de ácido sulfúrico, fumos brancos serão gerados; Use uma coifa bem ventilada.</li>
		<li>Manter a solução de reação no frasco durante a noite sem cobertura. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Extração deV DMDTA usando o método de extração de fase sólida (SPE)
	<ol>
<li>Após a reação de pé durante a noite, filtro da solução de reação utilizando uma seringa de C18 digite SPE baseada em sílica para aprisionar sintetizado DMDTAV sobre a resina. Atenção: Use uma coifa bem ventilada.</li>
		<li>Prepare o acetato de amônio 10 mM (pH 6.3) pela dissolução do acetato de amónio de 0,77 g em 1L de água desionizada. Eluir um volume suficiente de acetato de amónio de 10 mM através da seringa C18 (etapa 2.2.1) para extrair o adsorvida DMDTAV. Recolha o acetato de amónio filtrada em um copo de Petri (diâmetro interno mínimo de 160 mm) e altura mínima de 50 mm.</li>
		<li>Transferi o prato de petri de vidro em uma caixa de luva N2 atmosfera para impedir a transformação de espécies. Atenção: Certifique-se de solvente não é puxado para a bomba de vácuo através da saída da caixa de passagem.</li>
		<li>Secar até um precipitado branco de dimethyldithioarsinate é formado (cristalizada DMDTAV) sobre o prato de petri de vidro. Nota: O protocolo pode ser pausado aqui.</li>
	</ol>
</li>
	<li>
Verificação de armazenamento e sintetizado DMDTAV
	<ol>
<li>Pegue o precipitado branco de cristalizada DMDTAVe medir seu peso total, a medição de gravação. Atenção: Use uma coifa ventilada ou luvas para evitar a inalação de gás sulfeto de hidrogênio.</li>
		<li>Dissolver cristalizado DMDTAV em 50 mL de N2-água desionizada purgado em 50 mL de um tubo de centrifugação e filtrar qualquer precipitado através de um filtro de seringa 0,2 µm.</li>
		<li>Suponha que o total a partir DMA usadosV é convertido em DMDTAV, ou seja,≈2, 539 mg As· L-1. Diluir a solução-mãe DMDTAV para ≈1 mg· L-1 e ≈40 µg· L-1 para análise de verificação utilizando ESI-MS e HPLC-ICP-MS, respectivamente.</li>
		<li>Analise a m/z de DMDTAV utilizando ESI-MS11,16 e fragmentos, em m/z 171 no modo positivo-íon ou em m/z 169 no modo negativo-íon (tabela 1). Nota: Ver referência os valores de m/z (tabela 1).</li>
		<li>Analisar o cromatograma da DMDTAV na solução estoque usando HPLC-ICP-MS11,16,17 com condições adequadas de eluente e confirmar que um pico encontra-se no tempo de retenção, descrito em a literatura. Nota: Pureza de sintetizado DMDTAV deve ser calculada utilizando os resultados da análise da etapa 2.3.5.</li>
		<li>Analisar a concentração de arsênio total usando ICP-MS após digestão com ácido11 e calcular a concentração real de DMDTAV em solução-mãe de DMDTAV sintetizada usando fatores de diluição e pureza, como a seguinte equação: Total analisado como concentração (µg· L-1) · · Fator de diluição Pureza (%) = concentração de verdade DMDTAV DMDTAV em solução-mãe (µg· L-1)</li>
		<li>Armazenar a solução estoque de DMDTAV a 4 ° C no escuro por mais análise quantitativa especiação18.</li>
	</ol>
</li>
</ol>

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O protocolo desenvolvido clarificou passos críticos que estudos anteriores1,3,4,8,15 omitido ou abreviado, que pode ter levado a dificuldades com ou falha durante Síntese deV DMMTAV e DMDTA. Como DMMTAV é sensível à oxidação1,5, reagentes químicos para sua sínt...

Desde que o ácido dimethylarsinic (DMAV) demonstrou-se a expor tanto a toxicidade aguda e a genotoxicidade devido a sofrer metilação e thiolation após a ingestão de1,2, a via metabólica de arsênico thiolation tem foi intensamente estudado tanto in vitro e in vivo3,4 , bem como em meios ambientais (por exemplo, percolado de aterro)5,6. Estudos anteriores descobriram ambos reduziram e células thiolated análogos de DMAV na vida, por exemplo, dimethylarsinous ácido (DMAIII), ácido dimethylmonothioarsinic (DMMTAV) e dimethyldithioarsinic (ácido DMDTAV)7,8,9, com dimethylated thioarsenicals como DMMTAV apresentando toxicidade maior do que outro conhecido inorgânicos ou orgânicos arsenicals10. A abundância de thioarsenicals altamente tóxicos tem sérias implicações ambientais, uma vez que eles podem representar um risco para os seres humanos e o ambiente sob condições altamente sulfidic11. No entanto, os mecanismos de DMMTAV e DMDTAV (trans) formação e seus destinos em meios ambientais ainda exigem um estudo mais aprofundado. Assim, a análise quantitativa de thioarsenicals é necessário para melhorar a compreensão dos efeitos ambientais de DMMTAV e DMDTAV.
Embora produtos químicos padrão são o requisito-chave para análise quantitativa, os padrões de DMMTAV e DMDTAV são difíceis de obter, replicando a estudos anteriores, devido ao alto risco de transformação de espécies em outras espécies e procedimentos de síntese unstandardized12. Além disso, os métodos referenciados têm limitações que podem levar a dificuldades de ordem práticas em sintetizar os químicos padrão e realizar a análise quantitativa. DMMTAV DMDTAV comumente preparam e misturando DMAV, Na2S e H2para4 , em uma certa relação molar1 ou borbulhamento H2S de gás através de uma solução de DMAV 13,14. A substituição de recursos de método subida de oxigênio por enxofre usando um fornecimento directo de gás H2S, que é altamente tóxico e difícil de controlar para um usuário inexperiente. Por outro lado, o acima mistura método1, amplamente utilizado para a análise qualitativa de DMMTAV e DMDTAV em sudies ambiental5,6,12, apresenta o thiolation de DMAV com H2S gerados pela mistura Na2S e H2para que4 e produz DMMTAV e DMDTAV, permitindo mais fácil estequiométrico controle para produzir produtos químicos de alvo, como em comparação com o direto uso do gás de H2S.
A referência que mistura método procedimentos1,3,4,8,15 mencionado em limitações de exposição deste estudo em alguns dos seus passos críticos experimentais, que podem levar a falha experimental. Por exemplo, os detalhes da preparação de solvente específico (ou seja, água deionizada, água) e a extração e cristalização dos arsenicals sintetizados são excessivamente abreviados ou não descritas em detalhes suficientes. Tais dispersaram e informações limitadas sobre actos processuais podem levar à formação inconsistente de thioarsenicals e análise de quantificação confiável. Portanto, o protocolo modificado desenvolvido neste documento descreve a síntese de DMMTAV e DMDTAV soluções estoque com análise de separação quantitativa de espécies.

<table><tbody><tr><td>Cacodylic acid</td><td>Sigma-Aldrich</td><td>20835-10G-F</td><td></td></tr><tr><td>Sodium sulfide nonahydrate</td><td>Sigma-Aldrich</td><td>S2006-500G</td><td></td></tr><tr><td>Sulfuric acid 96%</td><td>J.T.Baker</td><td>0000011478</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A7262-500G</td><td></td></tr><tr><td>Formic acid 98%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>066-00461</td><td></td></tr><tr><td>Diethyl ether (Extra Pure)</td><td>Junsei Chemical</td><td>33475-0380</td><td></td></tr><tr><td>Adapter cap for 60 mL Bond Elut catridges</td><td>Agilent Technologies</td><td>12131004</td><td>Syringe type of SPE</td></tr><tr><td>Bond Elut C18 cartridge</td><td>Agilent Technologies</td><td>14256031</td><td>Syringe type of SPE</td></tr><tr><td>HyPURITY C-18</td><td>Thermo Scientific</td><td>22105-254630</td><td>5 um, 125 x 4.6 mm</td></tr><tr><td>Glovebox</td><td>Chungae-chun, Rep. of Korea</td><td></td><td>Customized&amp;nbsp;</td></tr><tr><td>Agilent 1260 Infinity Bio-inert LC</td><td>Agilent Technologies</td><td>DEAB600252, DEACH00245</td><td></td></tr><tr><td>Agilent Technologies 7700 Series ICP-MS</td><td>Agilent Technologies</td><td>JP12031510</td><td></td></tr><tr><td>Finnigan LCQ Deca XP MAX Mass Spectrometer System</td><td>Thermo Electron Corporation</td><td>LDM10627</td><td></td></tr></tbody></table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimethylated thioarsenicals, tais como o ácido dimethylmonothioarsinic (DMMTAV) e ácido dimethyldithioarsinic (DMDTAV), que são produzidas pela via metabólica de dimethylarsinic ácido (DMAV) thiolation, tem sido recentemente encontrado o ambiente, bem como os órgãos humanos. DMMTAV e DMDTAV podem ser quantificado para determinar os efeitos ecológicos de dimethylated thioarsenicals e sua estabilidade em meios ambientais. O método de síntese para esses compostos é unstandardized, fazendo replicando desafiador de estudos anteriores. Além disso, há uma falta de informação sobre técnicas de armazenamento, incluindo armazenamento de compostos sem transformação de espécies. Além disso, porque apenas informações limitadas sobre métodos de síntese estão disponíveis, pode haver dificuldades experimentais em sintetizar produtos químicos padrão e realizar a análise quantitativa. O protocolo apresentado neste documento fornece um método de síntese praticamente modificado para o thioarsenicals dimethylated, DMMTAV e DMDTAVe ajudará na quantificação da análise de separação de espécies usando líquido de alto desempenho cromatografia em conjunto com a espectrometria de massa de plasma indutivo (HPLC-ICP-MS). Os passos deste procedimento experimentais foram modificados, centrando-se na preparação de reagentes químicos, métodos de filtração e armazenamento.

Desde que DMMTAV foi erroneamente preparado pelo método de síntese a DMAIII 19, verificação de sintetizado DMMTAV DMDTAV é um passo fundamental para a síntese e a extração e a determinação do padrão ideal materiais químicos. Produtos químicos sintetizados podem ser verificados pelo pico de DMMTAV (154 MW g·mol-1) e proporção de massa-de-carga DMDTAV (170 MW g·mol-1</s...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Preparação de DMMTAV DMDTAV usando DMAV para aplicações ambientais: síntese, purificação e confirmação

Este artigo apresenta protocolos experimentais modificados por ácido dimethylmonothioarsinic (DMMTAV) e a síntese de ácido (DMDTAV) dimethyldithioarsinic, induzindo dimethylarsinic ácido (DMAV) thiolation através da mistura de DMAV , At2S e H2então4. O protocolo modificado fornece uma orientação experimental, desse modo, superando limitações as etapas de síntese que poderia ter causado falhas experimentais em análise quantitativa.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

preparation-dmmtav-dmdtav-using-dmav-for-environmental-applications

Research

JoVE Journal

Environment

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

5.5K visualizações.  Korea Basic Science Institute, Seoul Center. Este artigo apresenta protocolos experimentais modificados por ácido dimethylmonothioarsinic (DMMTAV) e a síntese de ácido (DMDTAV) dimethyldithioarsinic, induzindo dimethylarsinic ácido (DMAV) thiolation através da mistura de DMAV , At2S e H2então4. O protocolo modificado fornece uma orientação experimental, desse modo, superando limitações as etapas de síntese que poderia ter causado...

Vídeo: Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

Controlando o tamanho, forma e estabilidade de Polímeros Supramoleculares na Água

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engenharia

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Complexos de produção de transmembrana Disulfide-estabilizados Peptídicos para estudos estruturais

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Química

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Compactas pontos quânticos para uma única molécula de imagem

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Medição bioquímicos da Hipóxia Neonatal

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicina

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

preparação e<em&gt; In Vitro</em&gt; Caracterização de agentes de contraste à base de dendrímeros para Ressonância Magnética

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Química de Fluxo Contínuo: Reação de Difenildiazometano com Ácido p-Nitrobenzoico

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

Fabricação de superfícies reativas com escova, como filmes de quitosana de polímeros co bloco Azlactone-acrescida e

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Explorando o metiloma de arginina por espectroscopia de ressonância magnética nuclear

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Pesquisa do câncer

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Avaliação do estresse oxidativo em amostras biológicas usando o ensaio de substâncias reativas de ácido tiobarbitúrico

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

Detecção de trimetilamina em medicina derivada de animais por Headspace GC-MS

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Autor em destaque: Uma técnica aprimorada para detecção de trimetilamina em medicina derivada de animais por cromatografia gasosa headspace-espectrometria de massa quadrupolo em tandem  

Here, a headspace gas chromatography-tandem quadrupole mass spectrometry (HS-GC-MS/MS) method suitable for the determination of trimethylamine (TMA) in animal-derived medicines is described. The protocol includes sample pretreatment, headspace treatment, analysis conditions, methodological validation, and the determination of TMA in animal-derived medicines.

Uma Técnica Aprimorada para Detecção de Trimetilamina em Medicina Derivada de Animais por Cromatografia Gasosa Headspace-Espectrometria de Massas em Tandem Quadrupolo

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the ...

Preparação de DMMTA^V DMDTA^V usando DMA^V para aplicações ambientais: síntese, purificação e confirmação

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