Comparison of standard chromatographic procedures for the optimal purification of soluble human brain acetylcholinesterase.
With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak. Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2. Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis-à-vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS-PAGE of affinity-purified AChE revealed four major bands between 50 and 62 kDa, corresponding to the catalytic subunits of AChE as verified by an anti-AChE polyclonal antibody. A size-exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation-exchange using the state-of-the-art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.
Novales-Li
P
P
Department of Pharmacology, University of Oxford, UK.
D000110:Acetylcholinesterase / Q000134:cerebrospinal fluid / Q000302:isolation & purification*; D001921:Brain / Q000201:enzymology*; D002421:Caudate Nucleus / Q000201:enzymology; D002845:Chromatography / Q000379:methods*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification*; D011342:Procainamide; D012995:Solubility
D007527:Isoenzymes; D000110:Acetylcholinesterase; D011342:Procainamide