Name: dissolution dynamic nuclear polarization instrumentation for real-time enzymatic reaction rate measurements by nmr
Uploaded: 2016-02-23T15:00:00
Description: Watch this Scientific Journal Video about Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR at JoVE.com

Los autores agradecen al Dr. JJ van der Klink para la asistencia en la elección y el montaje de los equipos, así como el Dr. F. Kateb y el Dr. G. Bertho útil para los debates. CA fue apoyado por la Fundación Nacional de Ciencia de Suiza (subvención PPOOP2_157547). Reconocemos la financiación de Paris Sorbonne Cité (RMN @ Com, DIM Analytics, Ville de París, la Fundación de la Investigación Médica (FRM ING20130526708), y el Parteneriat Hubert Curien Brancusi 32662QK. Nuestro equipo es parte de los programas Equipex Paris-en-RESONANCIA y CACSICE.

NOTA: Todos los análisis de los datos se realizó utilizando el software comercial. 1. Preparar la solución polarizante <ol><li> Preparar 2 ml de una piruvato de sodio 1,12 M 13 C marcado con (Na + [CH 3 -CO- 13 COO] -, sustrato) solución dopa con 33 mM de radical TEMPOL (4-hidroxi-2,2,6,6 tetrametil-1-oxilo, agente polarizador) 4 en 2: 1 D 2 O / d 6 etanol durante 13 observaciones C. PRECAUCIÓN: Precauciones...

<ol>
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Los puntos críticos de la disolución experimento DNP RMN son: (i) el nivel de polarización conseguido por el sustrato, que determina la concentración de producto más baja necesaria para los experimentos, así como el número de adquisiciones de señal que se pueden realizar y (ii) los tiempos de vida de magnetización, en comparación con la duración de la transferencia entre la polarización y los sitios de detección y de la tasa de transformación de sustrato. El sistema de inyección de la configuración de DN...

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNP polarizer</td>
			<td style="width:287px;">Vanderklink s.a.r.l (Switzerland)</td>
			<td style="width:119px;">///</td>
			<td style="width:167px;">Cryostat and electronic equipment for sample polarization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum system components</td>
			<td style="width:287px;">Edwards vacuum (France)</td>
			<td style="width:119px;">Various</td>
			<td>
			- turbomolecular pumping setup
			- membrane pumping setup
			- high capacity roots pumping system
			- vacuum fittings and components
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNP 3.35T Magnet</td>
			<td style="width:287px;">Bruker (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">500MHz NMR Spectrometer</td>
			<td style="width:287px;">Bruker (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Origin 8.0</td>
			<td style="width:287px;">OriginLab (US)</td>
			<td style="width:119px;"></td>
			<td>Data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SODIUM PYRUVATE-1-13C, 99 ATOM % 13C</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">490709</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ETHANOL-D6, ANHYDROUS, 99.5 ATOM % D</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">186414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;4-Hydroxy-TEMPO 97%</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">176141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deuterium oxide</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">151882</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reduced nicotinamide adenine dinucleotide (NADH)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethylene-diaminetetraacetic acid (EDTA)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dithiothreitol (DTT)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phosphate buffer, pH&#160;=&#160;7.0</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LDH enzyme in&#160;</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;">L-2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin, BSA</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissolution dynamic nuclear polarization instrumentation for real-time enzymatic reaction rate measurements by nmr

La principal limitación de las investigaciones basados ​​en RMN es baja sensibilidad. Esto incita para largos tiempos de adquisición, evitando así que las mediciones de RMN en tiempo real de las transformaciones metabólicas. Hiperpolarización mediante la disolución DNP elude parte de la sensibilidad emite gracias a la gran magnetización nuclear fuera de equilibrio resultante de la transferencia de espín polarización de electrones al núcleo. La señal de RMN de alta obtenida puede ser utilizada para controlar las reacciones químicas en tiempo real. La desventaja de hiperpolarizado NMR reside en la ventana de tiempo limitado disponible para la adquisición de señales, que es generalmente del orden de la constante de tiempo de relajación longitudinal de espín nuclear, T 1, o, en casos favorables, en el orden de la constante de tiempo de relajación asociado con el singlete de estado de los núcleos acoplados, T LLS. La captación celular de moléculas endógenas y las tasas metabólicas puede proporcionar información esencial sobre el desarrollo tumoral y la respuesta al fármaco. Nurosos estudios de RMN hiperpolarizados anteriores han demostrado la relevancia de piruvato como sustrato metabólico para monitorizar la actividad enzimática in vivo. Este trabajo proporciona una descripción detallada de la configuración experimental y métodos necesarios para el estudio de las reacciones enzimáticas, en particular, la tasa de conversión de piruvato a lactato en presencia de lactato deshidrogenasa (LDH), por RMN hiperpolarizado.

ganancias de señal de RMN utilizando DNP disolución El efecto DNP consiste en la transferencia de la alta polarización de espines de los electrones no apareados, típicamente de moléculas de radicales estables, a los núcleos de RMN-activos, bajo irradiación con microondas de la muestra. Los radicales libres más usadas son de TAM (OXO63) y TEMPOL. 4 procedimientos de polarización utilizando TEMPOL pueden ser optimizados por &quot;polarización cruzada &#39;. <sup...

Watch this Scientific Journal Video about Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR at JoVE.com

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

Disolución dinámica de polarización nuclear Instrumentación para el tiempo real enzimática reacción de la tasa mediciones realizadas por RMN

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

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A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Análisis de la respiración en tiempo real mediante el uso de ionización secundaria Nanoelectrospray acoplada a espectrometría de masas de alta resolución

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Partícula magnética de ADN vinculante el análisis por la dispersión ligera dinámica y electroforética

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

U2O5 película preparación vía UO2 deposición por Sputtering de corriente directa y sucesiva oxidación y reducción con oxígeno atómico y el hidrógeno atómico

We describe a method to produce U2O5 films in situ using the Labstation, a modular machine developed at JRC Karlsruhe. The Labstation, an essential part ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Aspecto técnico de la síntesis automatizada y evaluación cinética en tiempo real de [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Niobium Oxide Films Deposited by Reactive Sputtering: Effect of Oxygen Flow Rate

Películas de óxido de niobio depositadas por sputtering reactivo: efecto de la tasa de flujo de oxígeno

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition ...