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Introduction Alzheimer’s disease (AD) is an irreversible, progressive brain disorder accounting for about 70 % of dementia cases. Worldwide, it means over 34 million patients suffering from loss of intellectual and social skills to such an extent that it affects their daily life [1]. Introduction of a cure for AD, however, is hindered by limited knowledge about its cause; currently available medications may only improve symptoms. Since amyloid plaques found in a brain tissue of AD patients cause the inflammatory reaction leading to degradation of neurons, specific inhibition of ß-secretase (BCE), enzyme responsible for formation of these plaques, appears to be a promising way for slowing down or even stopping the progression of the disease [2]. Capillary electrophoresis (CE) represents a suitable technique to determine the enzyme activity due to highly effective separations and minute sample consumption. Furthermore, a fused-silica capillary can also serve as a nanoliter-scale reaction vessel. Such procedures are referred to as on-line CE methods that integrate incubation of an enzymatic reaction, separation of reaction products, and their detection and quantitation into a single fully automated analysis. For these reasons, the main goal of this work was to develop an on-line CE method for studies of BACE activity. Experimental An Agilent 7100 CE system was used to perform all analyses. An uncoated fused-silica capillary (56 cm effective length; 75 ?m id) was thermostated at 37 °C. 5 % acetic acid was used as BGE. The capillary was rinsed with 50 % acetic acid for 1 min, deionized water for 1 min, 0.1 M NaOH for 1 min, deionized water for 1 min, and BGE for 2 min before every analysis. This procedure was sufficient to ensure good method repeatability despite a relatively high concentrations of BACE and model substrate, decapeptide SEVNLDAEFR, were injected into the capillary. The sample injection procedure consisted of the alternate hydrodynamic introduction of 4 plugs of the substrate solution (each injected for 3 s by 15 mbar) and 3 plugs of the BACE solution (each injected for 3 s by 30 mbar). Both solutions were prepared in 50 mM sodium acetate buffer (pH 4.25). The resulting reaction mixture was incubated for 12 min. After it, formed products were separated by concomitant application of 30 kV (465 V cm-1, positive polarity) and a positive pressure of 10 mbar. An on column UV-VIS detector set to 200 nm was employed for monitoring of DAEFR fragment (Fig. 1). Results A principle of transverse diffusion of laminar flow profiles (TDLFP) methodology was adopted for mixing inside the capillary because it is generic, robust, and rapid [3]. All parameters of the assay were optimized with respect to reaction products yields, their resolution, and method repeatability. The final method was then validated and used for on-line kinetic study of BACE. Results obtained were in a good agreement with conducted control off line study and published data determined using HPLC-MS method [4]. On the other hand, resulting values were for 2 orders of magnitude higher than published data determined using currently common FRET-based assays. This is probably given by limited solubility of fluorescent-labeled substrates which does not enable analysis of whole concentration range of the Michaelis-Menten kinetics. Regression analysis of the initial part of the curve and consecutive interpolation likely lead to systematic underestimation of the kinetic parameters. Additional studies employing other detection techniques will be therefore conducted in order to confirm these findings. Conclusion A method for on-line studies of BACE activity was developed. Utilization of soluble model substrate enabled thorough analysis of the enzyme’s reaction kinetics. Furthermore, due to generic nature of diffusion-based mixing, method is directly applicable for inhibition studies.
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