Choosing the right chromatographic support in making a new acetylcholinesterase-micro-immobilised enzyme reactor for drug discovery.
The aim of the present study was to optimize the preparation of an immobilized acetylcholinesterase (AChE)-based micro-immobilized enzyme reactor (IMER) for inhibition studies. For this purpose two polymeric monolithic disks (CIM, 3 mm x 12 mm i.d.) with different reactive groups (epoxy and ethylendiamino) and a packed silica column (3 mm x 5 mm i.d.; Glutaraldehyde-P, 40 microm) were selected as solid chromatographic supports. All these reactors were characterized in terms of rate of immobilization, stability, conditioning time for HPLC analyses, optimum mobile phase and peak shape, aspecific interactions and costs. Advantages and disadvantages were defined for each system. Immobilization through Schiff base linkage gave more stable reactors without any significant change in the enzyme behaviour; monolithic matrices showed very short conditioning time and fast recovery of the enzymatic activity that could represent very important features in high throughput analysis and satisfactory reproducibility of immobilization yield. Unpacked silica material allowed off-line low costs studies for the optimization of the immobilization step.
Bartolini
M
M
Dipartimento Di Scienze Farmaceutiche, Università Di Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
Cavrini
V
V
Andrisano
V
V
D000110:Acetylcholinesterase / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D015195:Drug Design; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D004800:Enzymes, Immobilized; D000110:Acetylcholinesterase