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Title:Analysis of non-classical neurotransmitters using capillary electrophoresis
Author(s):Cruz, LouAnn Latsnic
Doctoral Committee Chair(s):Sweedler, Jonathan V.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Neuroscience
Chemistry, Analytical
Abstract:Capillary electrophoresis (CE) is a powerful tool for the analysis of complex microenvironments and has found widespread applicability to studies in neuroscience. Depending upon the selectivity and sensitivity requirements of the particular application, a variety of detection schemes for CE are available. A review is presented of optical CE detectors.
The utility of CE for assessing the purity and stability of a pharmaceutical peptide, nisin, is investigated. With UV absorbance detection, degradants are not identified and unresolved components go undetected. Mass spectrometric analysis of isolated CE fractions provides molecular mass information that aids in degradant identification and determination of peak purity.
A CE method that enhances the ability to attach a fluorescent tag to low concentration peptide samples is developed that involves placing a solid phase material at the capillary inlet. Peptides are fluorescently derivatized on the material, eluted, electrophoretically separated, and detected downstream with laser-induced fluorescence.
The optimization of a CE separation method for the analysis of peptide neurotransmitters within cellular extracts and single neurons of a marine inveltebrate, Aplysia californica, is presented. UV absorbance is used to detect underivatized peptides at 10$\sp{-3}-10\sp{-5}$ M, while a post-column radionuclide phosphor-imaging detector is used to detect $\sp{35}$S-methionine radiolabeled peptides down to 10$\sp{-10}$ M.
A CE method utilizing direct UV absorbance detection that quantitates nitrite and nitrate levels within ganglia and individual neurons of the sea-slug, Pleurobranchaea californica, is described. The method correlates well to results obtained using NADPH-diaphorase staining, and can be used to monitor nitric oxide synthase activity and nitric oxide (NO) production in vivo.
NO is produced non-enzymatically from the reaction of arginine analogs with various reducing agents as elucidated by electron paramagnetic resonance (EPR). A CE method that quantitatively detects nitrite (an NO oxidation product) is developed that confirms the EPR results.
The nitroxyl anion, NO$\sp{-},$ one of the redox forms of NO, is extremely difficult to detect, due to its instability. A CE method that detects nitroxyl as NiNO(CN)$\sb3\sp{2-}$ is presented. The method may be useful for determining if nitroxyl is the active form of NO in a variety of biochemical reactions.
Issue Date:1996
Type:Text
Language:English
URI:http://hdl.handle.net/2142/22601
ISBN:9780591197846
Rights Information:Copyright 1996 Cruz, LouAnn Latsnic
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9712243
OCLC Identifier:(UMI)AAI9712243


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