高等植物光系统Ⅱ中强光照射产生超氧阴离子自由基的ESR探索

ESR Studies on the Superoxide Radicals Generated in Photosystem Ⅱ of Higher Plant

利用自旋捕捉电子顺磁共振 (ESR)的方法对从菠菜叶绿体中分离提纯的光系统Ⅱ (PSⅡ )颗粒产生O2 ·的机理进行了直接检测 .通过对样品充氧、加入超氧化物歧化酶 (SOD)抑制剂四氰乙烯 (TCNE)以及原位光照检测ESR信号等手段 ,在PSⅡ中检测到O2 ·与DMPO加合物的特征ESR信号 .而在没有SOD抑制剂的情况下 ,光照时PSⅡ中O2 ·与DMPO加合物浓度显著下降 .进一步实验发现PSⅡ中O2 ·产率与氧分子浓度直接正相关 .O2 ·产率还具有 pH值依赖性 ,在 pH值为 6 0～ 6 5范围内 ,O2 ·产率最高 ,大于此范围时则呈显著下降趋势 .而PSⅡ颗粒的Tris处理也将导致O2 ·产率的急剧减少 .以上结果证实水裂解放氧十分活跃的PSⅡ也是高等植物叶绿体在光照下产生活性O2 ·的主要部位 ,通常大部分的O2 ·能被内源SOD清除 ,且O2 ·的生成与PSⅡ的电子传递活性密切相关 .

Superoxide radicals(O 2 · ) produced in PSⅡ particles separated from spinach has been investigated by spin trapping electron spin resonance (ESR) technique. After bubbling with oxygen and incubating with tetracyanoethylene (TCNE) that acts as an inhibitor of superoxide dismutase (SOD) the experimental evidence of spin adduct of [DMPO O 2H] · can be obtained by in situ ESR measurements in PSⅡ particles. In contrast, adduct of O 2 · and DMPO produced under illumination is obviously decreased when SOD that usually performs as a scavenger of O 2 · is present. Furthermore, it is evident to find that the generation of O 2 · is positively correlated with the concentration of oxygen. The production of O 2 · is also pH dependent, and O 2 · concentration reaches maximum when pH is in the range of 6 0～6 5. Either high, or low pH value will lead it to descend steeply.In Tris HCl washed PSⅡ particles donor side ET inhibition can causes the production of O 2 · decreasing obviously. It can be concluded that the PSⅡ that is active in splitting water and oxygen generation is also a reactive site responsible for the photoinduced O 2 · generation in thylakoid of higher plant. In another word, the formation of O 2 · is relevantly correlated to the electron transport activity of PSⅡ.