Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method.
Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005959:Glucosidases / Q000302:isolation & purification*; D005962:Glucosylceramidase / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013194:Staining and Labeling
D005959:Glucosidases; D005962:Glucosylceramidase