Molecular recognition of cocaine by acetylcholinesterases for affinity purification and bio-sensing.
Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the cocaine derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the "affinity" bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 Umg(-1). The same cocaine derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.
Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Golm, Germany. knoesche@rz.uni-potsdam.de
Kristina
K
Knösche
Jan
J
Halámek
Alexander
A
Makower
Didier
D
Fournier
Frieder W
FW
Scheller
D000110:Acetylcholinesterase / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003042:Cocaine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry*; D004330:Drosophila; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004593:Electrophorus; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020099:Coated Materials, Biocompatible; C005618:benzoylecgonine; D000110:Acetylcholinesterase; D003042:Cocaine