An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases

A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases (EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of -15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K-m and K-i values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes. (C) 2011 Elsevier Inc. All rights reserved.