Short-end injection procedure in capillary electrophoresis for determination of enzymatic reaction kinetics

A capillary electrophoresis (CE) using short-end injection procedure has been developed for the determination of an enzymatic reaction kinetics. Indeed, the phosphodiesterase 3ʹ,5ʹ-cyclic nucleotide enzyme (PDE) hydrolyses cyclic adenosine 3ʹ,5ʹ-monophosphate (cAMP) to adenosine 5ʹ-monophosphate (AMP). Simultaneous separation of cAMP and AMP was performed with a short effective length of capillary (8.5 cm) between the regular capillary outlet and the detector. In order to get short run times, electroosmotic flow was also reversed by flushing fused-silica capillary with 0.2% aqueous solution of a cationic surfactant (hexadimethrin bromide) prior each run. In addition, the high ionic strength of electrophoretic buffer (40 mM KH2PO4, pH 7.5) improves detection sensitivity by stacking effect. Extremely short-effective length of capillary induces reduced analysis times (25 s rather than 3.5 min) and allows to monitor enzymatic activity by using directly the sample vial as catalysis reactor. Besides, CE presents the advantage of needing very low sample volumes (nl), so the determination of enzymatic reaction kinetics can be carried out without any reaction-medium perturbation or excessive amounts of enzyme.