A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates |
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Authors: | Dagmar Kratky Achim Lass Peter M Abuja Hermann Esterbauer Hartmut Kühn |
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Institution: | 1. Institute of Biochemistry, University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria;2. Institute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Strasse 3–4, 10115 Berlin, Germany |
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Abstract: | Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (Km=3.7 μM, kcat=17 s?1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (Km=0.64 μM, kcat=0.15 s?1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes. |
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Keywords: | Lipid peroxidation 15-Lipoxygenase Low-density lipoprotein Hydroperoxy lipid Chemiluminescence AAPH 2 2-azobis(2-amidinopropane) hydrochloride BHT butylated hydroxytoluene CE-OOH cholesteryl linoleate hydroperoxides CL chemiluminescence cps counts per second EDTA ethylenedinitrilotetraacetic acid HPLC high-performance liquid chromatography LDL low-density lipoprotein LOOH hydroperoxy lipids 15-LOX rabbit reticulocyte 15-lipoxygenase PBS phosphate-buffered saline PUFA polyunsaturated fatty acid SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis TMP tocopherol mediated lipid peroxidation |
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