Galland-Irmouli A V, Pons L, Luçon M, Villaume C, Mrabet N T, Guéant J L, Fleurence J
Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, EP CNRS 0616, Faculté de Médecine, BP 184, Vandoeuvre lès Nancy, France.
J Chromatogr B Biomed Sci Appl. 2000 Feb 28;739(1):117-23. doi: 10.1016/s0378-4347(99)00433-8.
Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample in liquid nitrogen, homogenisation in phosphate buffer and centrifugation. Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous elution system and detected by its pink colour and fluorescence. The pigment presented a typical spectrum of R-phycoerythrin, with three absorbance maxima at 499, 545 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A565/A280, a criterion for purity, was 3.2. A single protein of relative molecular mass 240,000 was detected on native-PAGE with silver staining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two major subunits with Mr 20,000 and 21,000, respectively, and a very minor subunit of Mr 30,000. These observations are consistent with the (alphabeta)6gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autumn and showed a good stability up to 60 degrees C and between pH 3.5 and 9.5. In conclusion, phycoerythrin of Palmaria palmata was purified in a single-step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria palmata will allow one to evaluate its fluorescence properties for future applications in biochemical techniques.
藻红蛋白是红藻和蓝细菌的一种主要捕光色素，被广泛用作荧光探针。在本研究中，通过将大型红藻掌状红皮藻样品在液氮中研磨、在磷酸盐缓冲液中匀浆并离心来提取藻红蛋白。然后使用连续洗脱系统的制备型聚丙烯酰胺凝胶电泳（PAGE）从该粗提物中纯化藻红蛋白，并通过其粉红色和荧光进行检测。该色素呈现出典型的R-藻红蛋白光谱，在499、545和565 nm处有三个吸光度最大值，在578 nm处有一个荧光最大值。吸光度比值A565/A280（纯度标准）为3.2。在天然PAGE上用银染法检测到一种相对分子质量为240,000的单一蛋白质。十二烷基硫酸钠-PAGE显示存在两个主要亚基，其相对分子质量分别为20,000和21,000，以及一个非常小的相对分子质量为30,000的亚基。这些观察结果与R-藻红蛋白的(αβ)6γ亚基组成特征一致。掌状红皮藻的藻红蛋白在秋季含量较高，在高达60摄氏度以及pH值在3.5至9.5之间时表现出良好的稳定性。总之，使用制备型PAGE一步法纯化了掌状红皮藻的藻红蛋白。获得掌状红皮藻的纯R-藻红蛋白将有助于评估其荧光特性，以便未来在生化技术中应用。
