Developing procedures for the large-scale purification of human serum butyrylcholinesterase.
Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.
Saxena
Ashima
A
Division of Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA. ashima.saxena@us.army.mil
Luo
Chunyuan
C
Doctor
Bhupendra P
BP
D001798:Blood Proteins / Q000737:chemistry*; D002091:Butyrylcholinesterase / Q000097:blood* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002852:Chromatography, Ion Exchange; D006801:Humans; D010949:Plasma / Q000737:chemistry*; D011342:Procainamide / Q000737:chemistry; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry
D001798:Blood Proteins; C032309:Cohn fraction IV; C035771:Sepharose CL 4B; D012685:Sepharose; D002091:Butyrylcholinesterase; D011342:Procainamide