Direct injection of pigment-protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC
Direct injection of pigment-protein complexes and membrane fragments suspended in water from phototrophs to C18 HPLC
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将来自光养生物的色素-蛋白质复合物和悬浮在水中的膜片段直接注射到 C18 HPLC
DOI：
10.1007/s11120-020-00735-w
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发表时间：
2020
影响因子：
3.7
通讯作者：
Harada Jiro
中科院分区：
文献类型：
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作者：
Takaichi Shinichi;Okoshi Akira;Otomo Seiu;Misumi Masahiro;Sonoike Kintake;Harada Jiro
我们发现，色素-蛋白质复合体、膜碎片和悬浮在水中的叶绿体中的色素包括类胡萝卜素和(细菌)叶绿素，可以直接进入C18高效液相色谱柱进行分析，而不需要任何其他处理。我们将该方法应用于紫色细菌的LH1-RC和色团、绿色硫磺细菌的叶绿体、蓝藻的类囊体膜以及菠菜的PSII和类囊体膜。高效液相洗脱图谱和色素组成与常规提取方法相同。这种方法的原理可能是，样品首先被捕获在柱的顶部，然后像往常一样，用高效液相色谱洗脱液立即提取色素并使用C18柱进行分离。在传统的提取方法中，首先用有机溶剂提取色素，然后挥发溶剂。然后将干燥的颜料溶解在有机溶剂中，过滤后进入C18高效液相色谱仪。这种方法的优点包括防止颜料异构化和氧化，以及可以注射所有样品。它的缺点包括变性蛋白质在柱顶积累，导致高效液相压力增加。使用防护柱可能会解决这个问题。样品、色谱柱和高效液相色谱系统等多种因素都会影响该方法的分析结果。尽管如此，我们认为一些样品可以用这种方法进行分析。
We discovered that pigments including carotenoids and (bacterio)chlorophylls in pigment–protein complexes, membrane fragments, and chlorosomes suspended in water could be injected directly into C18HPLC and analyzed without any other treatments. We applied this method to LH1-RC and chromatophores of purple bacteria, chlorosomes of green sulfur bacteria, thylakoid membranes of cyanobacteria, and PSII and thylakoid membranes of spinach. HPLC elution profiles and pigment composition were the same as those of the conventional extraction method. The principle of this method might be that samples are first trapped on top of column, followed by the immediate extraction of the pigments with the HPLC eluent and their separation using the C18column, as usual. In the conventional extraction method, pigments are first extracted with organic solvents, followed by evaporation of the solvents. The dried pigments are then dissolved in organic solvents and injected into C18HPLC after filtration. The advantages of this method include the preventions of pigment isomerization and oxidation and the possibility of injecting all samples. Its drawbacks include the accumulation of denatured proteins at the top of column, causing increased HPLC pressure. The use of a guard column might solve this problem. Many factors, such as samples, column, and HPLC systems, may affect this method. Nevertheless, we think that some samples can be analyzed using this method.