A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

Abstract

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 microA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.