Measuring nitric oxide in single neurons by capillary electrophoresis with laser-induced fluorescence: use of ascorbate oxidase in diaminofluorescein measurements

Analytical Chemistry
Won-Suk KimJonathan V Sweedler

Abstract

As a family of novel fluorescent indicators for nitric oxide (NO), the diaminofluoresceins (DAFs) have allowed real-time measurement of neuronal NO, an important gaseous neurotransmitter. However, the measurement of NO by the most commonly used NO sensor, 4,5-diaminofluorescein (DAF-2), is altered by two processes: the interaction of DAF-2 with intracellular dehydroascorbic acid (DHA) and the impact of ascorbic acid (AA) on the levels of N2O3, the intermediate product of the oxidation of NO that reacts with DAF-2. Similar AA/DHA effects are observed with other DAF probes, including DAF-FM and DAR-4M. To overcome these limitations, we use a specific enzymatic reaction to eliminate the confounding effect of AA on DAF quantitation of NO and then use capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to distinguish the various reaction products. First, the enzyme ascorbate oxidase (AO) is used to catalyze the oxidation of AA to DHA. Next, CE-LIF separates the fluorescent products of the reaction of DAF-2 with NO and DHA. Control experiments, including standard mixtures and single neurons with added NO donor, successfully demonstrate the utility of this approach. This protocol is further tested with homog...Continue Reading

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Citations

Aug 17, 2006·Analytical and Bioanalytical Chemistry·Imee G ArcibalAndrew G Ewing
May 20, 2008·Analytical Chemistry·Vratislav KostalEdgar A Arriaga
Jul 20, 2010·Annual Review of Analytical Chemistry·Evan M Hetrick, Mark H Schoenfisch
Jul 19, 2008·Annual Review of Analytical Chemistry·Laura M BorlandNancy L Allbritton
Jun 17, 2011·Annual Review of Biochemistry·Michael D PluthStephen J Lippard
Mar 15, 2012·Analytical and Bioanalytical Chemistry·Dulan B GunasekaraJosé Alberto Fracassi da Silva
Sep 7, 2011·Plant Science : an International Journal of Experimental Plant Biology·Luis A J MurElena Prats
Oct 6, 2009·Analytica Chimica Acta·Maura PerryRobert T Kennedy
Apr 14, 2009·Analytical Biochemistry·Jianguo Fang, Richard B Silverman
Dec 18, 2007·Journal of Neuroscience Methods·Xiaoying YeJonathan V Sweedler
Dec 27, 2008·Journal of Immunological Methods·Adriana TiscorniaAlfonso Cayota
Jun 9, 2007·Electrophoresis·Kimia SobhaniNorman J Dovichi
Mar 28, 2008·The Analyst·Xiaoying YeJonathan V Sweedler
Jan 26, 2010·The Analyst·Zheng ShenStephen J Haswell
Jan 1, 2015·American Journal of Physiology. Lung Cellular and Molecular Physiology·Shuai YuanChristopher G Kevil
Sep 11, 2020·Analytical Methods : Advancing Methods and Applications·Jay Sibbitts, Christopher T Culbertson
May 23, 2008·Analytical Chemistry·Mark LowryIsiah M Warner
Jun 10, 2010·ACS Chemical Neuroscience·Xiaoying YeJonathan V Sweedler
Aug 29, 2019·ACS Omega·Carles Felip-LeónFrancisco Galindo

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