普通离子交换剂用于HPLC柱层析法快速分离眼镜王蛇毒(英文)

Rapid and Small Scale Isolation of the Toxins from Guangxi King Cobra(Ophiophagus hannah)Venom Using Ion-Exchange HPLC

目的 为了经济快速分离眼镜王蛇(Ophiophagushannah,Oh)蛇毒中的毒素成分。 方法 用普通离子交换剂于高效液相色谱柱 (HPLC) TSKgel SP-Toyopearl 65 0 SF (4× 1 5 0 mm)层析法 ,实验取得最佳分离条件后 ,将蛇毒样品上柱后进行梯度洗脱 ,各洗脱峰收集后在 Cosmosil 5 C4-AR-3 0 0柱 (4 .6× 1 5 0 mm)上进行逆相 HPLC分析。非单峰组分再进行 HPLC凝胶过滤柱TSKgel Toyopearl HW-40 Fine(4× 2 5 0 mm)层析 ,层析峰组分再进行 HPLC逆相分析。 结果 眼镜王蛇毒经HPLC离子交换柱层析获得了 1 6个蛋白组分 ,其中有 5个组分经逆相 HPLC分析单一组分 ;另外的复合性组分再进行 HPLC凝胶过滤柱层析后又得到 5个单峰蛋白组分。 结论 HPLC离子交换柱层析对分离蛇毒蛋白很有实用价值 ,特别是蛇毒样品量少的情况下 (1 0 ug)也能较好分离。还具有分离时间短 (1 h左右 ) ,无须低温条件等优点。

For rapid isolation and analysis of the toxic factors in the venom of king cobra(Ophiophagus hannah,Oh)We used the technique of high pressure liquid chromatography (HPLC) with an ion exchange column of TSKgel SP Toyopearl 650 Superfine (4 × 150 mm) The better isolating conditions of absorbence,elution buffer (pH,solute ionic strength),gradient schedule and flow rate were examined and determined The absorbence at wavelength of 230 nm was the best condition for determine the concentration proteins of Oh venom in ammonium acetate buffer On ion exch age HPLC,proteins in Oh venom were separated in a gradient manner from 20 mM to 0 2 M (and pH,from 5 8 to 8 0) at a flow rate of 0 3 ml/min at room temperature (25℃ ) After the crude venom was injected to HPLC,16 peaks of proteins were obtained On the analysis of the separated peak using reverse phase HPLC on a Cosmosil 5C4 AR 300 (4 6 mm× 150 mm) column,5 peaks among them were shown as a single peak Gel permeation HPLC on a TSKgel Toyopearl HW 40 Fine column was used for further purification of the fractions which had not shown as a single peak on reverse phage HPLC,and another five fractions of a single protein were obtained The results showed this ion exchange column could be valuable for rapid fractionation of Oh venom,especially in a small scale The gel permeation HPLC also could be useful for further purifying the complex fractions which could not be separated by ion exchange HPLC