通过pH峰聚焦逆流色谱法从粗制牛心提取物中纯化乳酸脱氢酶。
Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography.
作者信息
Shibusawa Yoichi, Misu Naomi, Shindo Heisaburo, Ito Yoichiro
机构信息
Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 5;776(2):183-9. doi: 10.1016/s1570-0232(02)00348-3.
pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.
采用pH峰聚焦逆流色谱法（CCC），使用交叉轴螺旋行星离心机（CPC）从粗制牛心提取物中纯化乳酸脱氢酶（LDH）。实验采用两组聚合物相系统，分别由16%（w/w）聚乙二醇（PEG）1000 - 12.5%（w/w）磷酸钾缓冲液和15%（w/w）PEG 1540 - 15%（w/w）硫酸铵组成，各系统处于不同pH值。在PEG 1540 - 硫酸铵聚合物相系统中，通过在上层固定相中添加保留剂（10 mM乙酸）和在下层流动相中添加洗脱剂（100 mM氢氧化钠），获得了最佳结果。在流速为0.5 ml/min时，LDH以尖锐峰的形式洗脱出来，与其他蛋白质得到了很好的分离。通过LDH酶活性以及十二烷基硫酸钠 - 聚丙烯酰胺平板凝胶电泳对收集的馏分进行分析，以检测污染的蛋白质。LDH直接从粗制牛心提取物中以浓缩状态纯化出来。
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