Proteins damaged by free-radical-generating systems in the presence of oxygen yield relatively long-lived protein hydroperoxides. These hydroperoxides have been shown by e.p.r. spectroscopy to be readily degraded to reactive free radicals on reaction with iron(II) complexes. Comparison of the observed spectra with those obtained with free amino acid hydroperoxides had allowed identification of some of the protein-derived radical species (including a number of carbon-centred radicals, alkoxyl radicals and a species believed to be the CO2 radical anion) and the elucidation of novel fragmentation and rearrangement processes involving amino acid side chains. In particular, degradation of hydroperoxide functions on the side chain of glutamic acid is shown to result in decarboxylation at the side-chain carboxy group via the formation of the CO2 radical anion; the generation of an identical radical from hydroperoxide groups on proteins suggests that a similar process occurs with these molecules. In a number of cases these fragmentation and rearrangement reactions give rise to further reactive free radicals (R., O2-./HO2., CO2-.) which may act as chain-carrying species in protein oxidations. These studies suggest that protein hydroperoxides are capable of initiating further radical chain reactions both intra- and inter-molecularly, and provide information on some of the fundamental mechanisms of protein alteration and side-chain fragmentation.

The mechanisms of formation and the nature of the altered amino acid side chains formed on proteins subjected to oxidant attack are reviewed. The use of stable products of protein side chain oxidation as potential markers for assessing oxidative damage in vivo in humans is discussed. The methods developed in the authors laboratories are outlined, and the advantages and disadvantages of these techniques compared with other methodologies for assessing oxidative damage to proteins and other macromolecules. Evidence is presented to show that protein oxidation products are sensitive markers of oxidative damage, that the pattern of products detected may yield information as to the nature of the original oxidative insult, and that the levels of oxidized side-chains can, in certain circumstances, be much higher than those of other markers of oxidation such as lipid hydroperoxides.

Pseudomonas aeruginosa alginate was purified and characterized in terms of uronic acid, carbohydrate and protein content, as well as by infra-red spectroscopy and gel electrophoresis. Added exogenous bacterial alginate inhibited the uptake and degradation of both viable and non-viable radiolabelled non-mucoid P. aeruginosa by resident mouse peritoneal macrophages. Alginic acid (from seaweed) inhibited the same parameters to almost the same degree. Bacterial alginate also inhibited the uptake of fluorescent-labelled zymosan and latex particles. Starch, at equivalent viscosity to the alginate, inhibited the uptake and degradation of radiolabelled nonviable P. aeruginosa to a greater extent, but Dextran T500 had no effect. This suggests that the viscous nature of alginate exerts a non-specific inhibitory effect on the uptake and subsequent degradation of phagocytosible particles.

Cholesterol efflux to apolipoprotein A-1 (apoA-1) from cholesterol-loaded macrophages is an important anti-atherosclerotic mechanism in reverse cholesterol transport. We recently provided kinetic evidence for two distinct pathways for cholesterol efflux to apoA-1 [Gaus et al. (2001) Biochemistry 40, 9363]. Cholesterol efflux from two membrane pools occurs sequentially with different kinetics; a small pool rapidly effluxed over the first hour, followed by progressive release from a major, slow efflux pool over several hours. In the present study, we propose that the rapid and slow cholesterol efflux pools represent cholesterol derived from lipid raft and nonraft domains of the plasma membrane, respectively. We provide direct evidence that apoA-1 binds to both lipid raft and nonraft domains of the macrophage plasma membrane. Conditions that selectively deplete plasma membrane lipid raft cholesterol, such as incorporation of 7-ketocholesterol or rapid exposure to cyclodextrins, block apoA-1 binding to these domains but also inhibit cholesterol efflux from the major, slow pool. We propose that cholesterol exported to apoA-1 from this major slow efflux pool derives from nonraft regions of the plasma membrane but that the interaction of apoA-1 with lipid rafts is necessary to stimulate this efflux.

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