Lipoprotein oxidation is considered a key event in the development of atherosclerotic disease. Low-density lipoprotein (LDL) is a highly compartmentalized system consisting of phospholipids in the outer layer, with cholesteryl esters in the core, apolipoprotein B, vitamin E, ubiquinone, -carotene, etc. being distributed throughout the particle. In order to fully understand the mechanism of protection afforded by antioxidants against LDL oxidation, it is necessary to monitor radical reactions in the outer monolayer as well as in the protein and core lipid, We have used the ESR spin-labeling technique to monitor radical reactions in LDL. This involves covalent labeling of cysteine and lysine residues in LDL using different spin probes. The extent of labeling is dependent on the concentration of the spin probes and the time of incubation. Using chromium oxalate or nickel chloride as paramagnetic relaxing agents, we have determined the location of spin-labeling sites. We show that lysine- and cysteine-labeled nitroxides are destroyed to varying degrees during Cu2+-initiated oxidation of LDL. The profile for the disappearance of nitroxide is similar to LDL peroxidation and is dependent on BHT concentration. Using this methodology, we can monitor radical reactions at specific locations in LDL.
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