Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography.
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.
Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. sibusawa@ps.toyaku.ac.jp
Yoichi
Y
Shibusawa
Naomi
N
Misu
Heisaburo
H
Shindo
Yoichiro
Y
Ito
D000818:Animals; D002417:Cattle; D003377:Countercurrent Distribution / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*
D007770:L-Lactate Dehydrogenase