Davey M W, Lambein F
Laboratorium voor Fysiologische Scheikunde, Rijksuniversiteit Gent, Belgium.
Anal Biochem. 1992 Nov 1;206(2):226-30. doi: 10.1016/0003-2697(92)90357-d.
Procedures are described for the quantitative, semi-preparative isolation of individual cyanobacterial heterocyst-type glycolipids (HGs) by reverse-phase HPLC (RP-HPLC) and the modifications to conventional techniques necessary to prevent significant HG losses during sample preparation. Total lipids are obtained from cultures of nitrogen-fixing cyanobacteria by triplicate extraction with 200 packed-cell volumes of chloroform/methanol (1/1, v/v), filtered, and redissolved in chloroform for loading onto a short disposable column of acid-washed silica. After removal of neutral lipids, pigments, and the majority of monogalactosyldiacylglycerol with chloroform and chloroform/acetone, HGs are eluted along with other complex lipids in methanol. The complex lipids are then fractionated by TLC and the HGs isolated as two classes of differing mobilities. The individual components of each class are then resolved by isocratic elution with methanol/water (91/9, v/v) from a C18 RP-HPLC column with refractive index detection. Samples of up to approximately 1.0 mg lipid can be completely separated and the major components isolated in high purity from a single run. Structural studies on the major HG of Nostoc azollae show it to be the glycosylated hexacosane-1,3,25-triol found by others in Anabaena cylindrica.
本文描述了通过反相高效液相色谱（RP-HPLC）对单个蓝藻异形胞型糖脂（HG）进行定量、半制备分离的方法，以及为防止样品制备过程中HG大量损失而对传统技术进行的改进。通过用200个填充细胞体积的氯仿/甲醇（1/1，v/v）进行三次萃取，从固氮蓝藻培养物中获得总脂质，过滤后再溶解于氯仿中，用于加载到短的一次性酸洗硅胶柱上。在用氯仿和氯仿/丙酮去除中性脂质、色素和大部分单半乳糖基二酰甘油后，HG与其他复合脂质一起在甲醇中洗脱。然后通过薄层层析（TLC）对复合脂质进行分离，将HG分离为两类迁移率不同的脂质。然后通过在配备折光率检测器的C18 RP-HPLC柱上用甲醇/水（91/9，v/v）等度洗脱，分离每类中的各个组分。高达约1.0 mg脂质的样品可以完全分离，并且主要成分可以从单次运行中以高纯度分离出来。对满江红鱼腥藻主要HG的结构研究表明，它是其他人在圆柱鱼腥藻中发现的糖基化十六烷-1,3,25-三醇。
