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目的:建立蛇毒血凝酶纯度测定方法。方法:分别采用SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)和等电聚焦法(IEF),对凝胶考染并扫描;采用体积排阻高效液相色谱法(SEC-HPLC),使用TSK G3000SW(7.8 mm×300 mm)凝胶柱,流动相为55 mmol.L-1柠檬酸钠-0.01%Tween 80溶液;流速0.5 mL.min-1,检测波长280 nm;采用反相高效液相色谱法(RP-HPLC),使用C8Vydac(4.6 mm×250 mm,5μm)色谱柱,流动相为0.1%三氟乙酸溶液(A)-含0.1%三氟乙酸的90%乙腈(B),梯度洗脱,流速0.8 mL.min-1,;柱温为45℃,检测波长:280 nm。结果:4种方法测得蛇毒血凝酶的纯度分别为89.3%,88.5%,95.4%,87.4%。结论:对于蛇毒血凝酶纯度测定,非还原SDS-PAGE、IEF及RP-HPLC法均可准确地反映其样品纯度。
Objective: To establish a method for determining the purity of snake venom hemagglutinin. Methods: The gel was detected and scanned by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) respectively. The inhibitory activity of the gel was detected by size exclusion high performance liquid chromatography Using TSK G3000SW (7.8 mm × 300 mm) gel column, the mobile phase was 55 mmol·L-1 sodium citrate-0.01% Tween 80 solution, the flow rate was 0.5 mL.min-1 and the detection wavelength was 280 nm. The mobile phase consisted of 0.1% trifluoroacetic acid (A) - 90% acetonitrile (B) with 0.1% trifluoroacetic acid using liquid chromatography (RP-HPLC) using a C8 Vydac (4.6 mm × 250 mm, , Gradient elution, flow rate 0.8 mL.min-1 ,; column temperature 45 ℃, detection wavelength: 280 nm. Results: The purity of venom hemagglutinin measured by four methods were 89.3%, 88.5%, 95.4% and 87.4%, respectively. Conclusion: The purity of snake venom hemagglutinin, non-reducing SDS-PAGE, IEF and RP-HPLC can all accurately reflect the purity of the sample.