Takenaka Y, Miki M, Yasuda H, Mino M
Department of Pediatrics, Osaka Medical College, Japan.
Arch Biochem Biophys. 1991 Mar;285(2):344-50. doi: 10.1016/0003-9861(91)90370-x.
Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.
偶氮化合物使我们能够通过热分解以恒定速率在所需位点产生过氧自由基，也就是说，水溶性化合物在水相中产生引发自由基，而脂溶性化合物在膜脂层内引发氧化反应。利用在不同位点产生的这些自由基，我们氧化红细胞血影膜，以研究α-生育酚消耗、脂质过氧化引发与蛋白质损伤之间的关系。当在水相中产生自由基时，膜蛋白硫醇的损失与膜生育酚的消耗同时出现，并且在生育酚耗尽后膜脂质发生过氧化。另一方面，当在脂质区域引发自由基时，硫醇的氧化和硫代巴比妥酸反应性物质的形成受到抑制，从而在生育酚降至临界水平以下之前出现诱导期。我们的结果表明，外在蛋白的表面硫醇可能与α-生育酚竞争捕获水相自由基，并在一定程度上使生育酚得以保留，而内在埋藏硫醇的氧化可能由于生育酚消耗后产生的脂质衍生自由基而开始。总之，膜中的α-生育酚可以有效地中断自由基链以抑制脂质过氧化。然而，生育酚对抑制膜蛋白损伤的作用，表现为硫醇的损失和高分子量蛋白质的形成，会因初始自由基产生的位点不同而有所差异。
