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Degree

Master of Science, Miami University, Chemistry, 2007.

Abstract

Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of only isolated double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent detection of liposomes and mitochondria through its interaction with CL, specific quantitative determination of CL in solution using NAO is uncommon. In this work, we have developed a fluorescence quenching method for CL using NAO. The optimum excitations are 499 and 518 nm at low and high NAO concentration, respectively. The optimum emissions are varied from 518 to 530 nm. The interaction of sodium n-dodecyl sulfate (SDS), sometimes used for lipid extraction from cells, and other PLs such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethnolamine (PE), phosphatidylglycerol (PG), phosphatdiylserine (PS), sphingomyelin (SM) and cholesterol, with NAO is investigated. The fluorescence intensity of 0.5 µM NAO signal is strongly quenched by SDS below 25% methanol in water. With a methanol content above 50%, no quenching of NAO by SDS or the PLs with the exception of PG above 8 µM is observed. Using 50-50 methanol-water, the fluorescence as a function of reaction time for the NAO-CL interaction is quite stable from 3 to at least 15 min. Concentrations of 5, 10, 20, and 50 µM NAO are considered and 20 µM NAO provides a linear fluorescence response from 0.2 – 10 µM CL. The detection limit is 0.2 µM and the limit of quantification is 0.6 µM. Acridine orange (AO) and phenosafranin (PSF) dyes are less effective as fluorescent probes for CL. CL in whole cell and membrane samples is quantitatively determined by standard addition to be in the 0.2-1.5 µM range. The increase of CL as compared to the controls is not significantly different in all samples subjected to cell death using staurosporine.

Subject Headings

Chemistry, Analytical

Keywords

Fluorescence; Cardiolipin; Phospholipids; 10-Nonyl acridine orange

Advisor

Neil D Danielson

Pages

85p.

Document number: miami1169691746
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=miami1169691746

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