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

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