Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography.
Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.
Shibusawa
Yoichi
Y
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
Fujiwara
Takiko
T
Shindo
Heisaburo
H
Ito
Yoichiro
Y
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry*; D003377:Countercurrent Distribution / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008099:Liver / Q000201:enzymology*
D004396:Coloring Agents; D008024:Ligands; D000426:Alcohol Dehydrogenase