Capillary Electrophoretic Enzyme Assays

Abstract

In the past years, capillary electrophoresis has become a frequently used technique for enzyme assays due to the high separation efficiency and versatility as well as small sample size and low consumption of chemicals. The capillary electrophoresis assays can be divided into two general categories: pre-capillary (or offline) assays and in-capillary (or online) assays. In pre-capillary assays, the incubation is performed offline and substrate(s) and product(s) are subsequently analyzed by capillary electrophoresis. In in-capillary assays enzyme reaction and separation of the analytes are performed inside the same capillary. In such assays the enzyme is either immobilized or in solution. The latter techniques is also referred to as electrophoretically mediated microanalysis (EMMA) indicating that the individual steps of the incubation as well as analysis are performed via electrophoretic phenomena. This chapter describes both techniques using the deacetylation of acetyl-lysine residues in model peptides by sirtuin enzymes as well as the hydrolysis of acetylthiocholine by acetylcholinesterase as examples.

Recent advances in CE‐mediated microanalysis for enzyme study

“…In order to avoid protein adsorption onto the capillary wall or even blocking of the capillary, deproteinization prior to sample injection can be achieved by addition of ACN in the quenching step of the reaction followed by centrifugation of the precipitated proteins. A stacking effect can be simply achieved in the precapillary assay by the injection of low ionic strength samples that can be obtained using ACN for quenching of the reaction . Protein adsorption to the capillary wall may be reduced using permanently polymer‐coated capillaries or employing a dynamic coating of the capillary wall .…”