For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X‐100. Subsequent sucrose density gradient centrifugation resulted in three or four red‐colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in‐gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

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用于分离甲藻 Oxyrrhis marina 蛋白视紫红质的简单方案

首次分离出海洋甲藻 Oxyrrhis marina 的天然蛋白视紫红质。将总细胞膜部分在珠子搅拌器中切碎并用洗涤剂 Triton X-100 溶解。随后的蔗糖密度梯度离心产生了三个或四个红色条带。未溶解但仍呈红色的膜沉淀在底部。对于这些波段中的每一个，吸光度最大值记录在大约 514-516 nm 处，肩部朝向较短的波长（470-490 nm）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示最上面的条带代表游离的视网膜发色团，因为它不含蛋白质。其他条带几乎是纯蛋白视紫红质级分，因为条带模式显示了一种 25 kDa 的主要蛋白质。胰蛋白酶，25 kDa 蛋白质和高于和低于 25 kDa 的微弱蛋白质条带的凝胶内消化随后进行质谱分析，证实这些蛋白质条带几乎完全由视紫红质蛋白组成。仅检测到少数其他蛋白质的单个肽。总共，至少有七个预测的视紫红质蛋白序列得到了实验验证。