Development and characterization of beta-secretase monolithic micro-immobilized enzyme reactor for on-line high-performance liquid chromatography studies.
beta-Site APP cleavage enzyme 1 (BACE-1) is a transmembrane aspartyl protease that cleaves the amyloid-beta precursor protein (APP), which is abundant in neurons. BACE-1 is required for the generation of amyloid-beta (Abeta) peptides implicated in the pathogenesis of Alzheimer's disease (AD). It is widely believed that halting the production of Abeta peptide, by inhibition of BACE-1, is an attractive therapeutic modality for the treatment of Alzheimer's disease. BACE-1 has never been immobilized before. In the present study, for the first time, human recombinant beta-secretase micro-immobilised enzyme reactor (hrBACE-1-micro-IMER) was prepared by using an in situ immobilisation procedure on an ethylendiamine monolithic convective interaction media (EDA-CIM) disk. The activity and kinetic parameters of the hrBACE-1-micro-IMER were investigated by insertion in a HPLC system with fluorescent and mass detection. The micro-IMER was characterized in terms of units of immobilised hrBACE-1 and best mobile phase conditions for activity, by using as substrate casein-FITC and JMV2236, a peptide mimicking the Swedish-mutated APP (amyloid precursor protein) sequence. The characterization of the hrBACE-1-micro-IMER in terms of number of enzymatic active units after covalent linking to the solid matrix was performed by using the JMV2236 peptide as substrate in a HPLC-MS system. JMV2236 was injected into the hrBACE-1-micro-IMER and enzymatically cleaved; the product of the enzymatic cleavage and the remaining non-cleaved substrate were collected on a C18 column trap and switched to the LC-electrospray ionization MS system for kinetic constants determination. Inhibition studies were carried out. The effect of donepezil and pepstatin A, as BACE-1 inhibitors, was evaluated by simultaneous injection of the compounds with the peptidic substrate. The relative IC(50) values were found in agreement with that derived by the conventional fluorescence method, confirming the applicability of this new IMER for on-line inhibition studies. The main advantages of the hrBACE-1-micro-IMER approach over the conventional methods were found to be the increased enzyme efficiency, stability and the decreased time of analysis.
Mancini
Francesca
F
Department of Pharmaceutical Sciences, Via Belmeloro 6, University of Bologna, 40126 Bologna, Italy.
Naldi
Marina
M
Cavrini
Vanni
V
Andrisano
Vincenza
V
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000077265:Donepezil; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007189:Indans / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D002364:Caseins; D004800:Enzymes, Immobilized; D007189:Indans; D010436:Pepstatins; D010880:Piperidines; D000077265:Donepezil; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C031375:pepstatin