Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

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外文摘要：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.

外文关键词：

pH-peak focusingcounter-current chromatographylactic acid dehydrogenase

作者：

Yoichi Shibusawa、Naomi Misu、Heisaburo Shindo、Yoichiro Ito

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作者单位：

Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA

出版年：

2002

Journal of Chromatography, Biomedical Applications

ISSN：0378-4347

年,卷(期)：2002.776(2)