Laboratory of Biophysics, Department of Experimental Physics, Faculty of Science, Palacky University, 77146 Olomouc, Czech Republic.
Free Radic Biol Med. 2010 Jan 15;48(2):264-74. doi: 10.1016/j.freeradbiomed.2009.10.051. Epub 2009 Oct 30.
Electron paramagnetic resonance (EPR) spin trapping spectroscopy is an important method used in free radical research; however, its application in biological systems is hindered by EPR silencing of spin adducts. Previous studies in superoxide-generating chemical systems have shown that spin adducts can be partially stabilized by cyclodextrins. In this work, for the first time, this proposed protective effect of cyclodextrins is investigated in a real biological sample-in isolated thylakoid membranes and photosystem II (PSII) particles with EMPO as a spin trap. It is shown that (i) randomly methylated beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin form inclusion complexes with EMPO-superoxide adducts (EMPO-OOH), (ii) both cyclodextrins increase the intensity of the EMPO-OOH EPR signal in PSII particles up to five times, (iii) higher EMPO-OOH EPR signal intensity is a result of increased stability of EMPO-OOH, and (iv) the extent of the protection of EMPO-OOH adduct provided by cyclodextrins is different in thylakoids and PSII particles. Along with the spin trapping data, the toxicity of cyclodextrins is also discussed with particular focus on photosynthetic preparations. The presented data show that both tested cyclodextrins can be used as valuable tools to improve the sensitivity of spin trapping in biological samples.
电子顺磁共振（EPR）自旋捕获光谱学是自由基研究中一种重要的方法；然而，其在生物系统中的应用受到 EPR 对自旋加合物的沉默的阻碍。先前在超氧化物生成化学系统中的研究表明，环糊精可以部分稳定自旋加合物。在这项工作中，首次在真实的生物样品中——以 EMPO 作为自旋捕集剂的分离类囊体膜和光系统 II（PSII）颗粒中，研究了环糊精的这种预期保护作用。结果表明：（i）随机甲基-β-环糊精和 2-羟丙基-β-环糊精与 EMPO-超氧自由基加合物（EMPO-OOH）形成包合物；（ii）两种环糊精使 PSII 颗粒中 EMPO-OOH 的 EPR 信号强度增加了五倍；（iii）更高的 EMPO-OOH EPR 信号强度是由于 EMPO-OOH 的稳定性增加所致；（iv）环糊精对 EMPO-OOH 加合物的保护程度在类囊体和 PSII 颗粒中是不同的。除了自旋捕获数据外，还特别关注光合制剂，讨论了环糊精的毒性。所呈现的数据表明，两种测试的环糊精都可以用作提高生物样品中自旋捕获灵敏度的有价值工具。
