Yu Daoyong, Huang Guihong, Xu Fengxi, Ge Baosheng, Liu Shuang, Xu Hai, Huang Fang
State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, 266580, Shandong, China,
Photosynth Res. 2014 Nov;122(2):203-13. doi: 10.1007/s11120-014-0022-8. Epub 2014 Jun 20.
Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-D-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of (1)O2, such as 1 mM NaN3, may be added into the mixture.
表面活性剂在体外对光系统I(PSI)的增溶作用中发挥着重要作用。使用 Triton X-100(TX)、正十二烷基-β-D-麦芽糖苷(DDM)和十二烷基硫酸钠(SDS)在MES缓冲液中增溶PSI颗粒,以比较表面活性剂及其用量对PSI表观耗氧率的影响。通过结合蔗糖密度梯度离心、非变性聚丙烯酰胺凝胶电泳(Native PAGE)和77K荧光以及表观耗氧情况,分析了表面活性剂增强PSI表观耗氧活性的本质。聚集的PSI颗粒可被表面活性剂分子分散成胶束,表面活性剂增溶的PSI的表观耗氧率高于完整的PSI颗粒。对于DDM,PSI颗粒大多以完整的三聚体形式被增溶。对于TX,PSI颗粒以不完整的三聚体和一些单体形式被增溶。对于更强的表面活性剂SDS,PSI颗粒完全以单体及其亚基形式被增溶。耗氧率的增强不能仅用表面活性剂对增溶PSI单体和三聚体形式之间平衡的影响来解释。当通过基于耗氧的方法评估PSI的电子转移活性时必须谨慎,因为表观耗氧率受PSI中解偶联叶绿素(Chl)的影响,即解偶联Chl的量越大,表观耗氧率越高。77K荧光光谱可用于确保系统中不存在解偶联Chl。为了消除微量解偶联Chl的影响,可向混合物中加入一种有效的(1)O2物理猝灭剂,如1 mM叠氮化钠。