A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase

Abstract

In the current study, capillary electrophoresis (CE)-based enzyme assay for characterization and inhibition study of bovine carbonic anhydrase II (bCA II) was developed. The developed method is the first CE assay for carbonic anhydrase (CA). The method was optimized in order to get short analysis time, minimal sample volume consumption, and high resolution of substrate and product. The CE conditions were optimized as follows: fused-silica capillary (30cm effective length×75μm i.d.), pressure injection for 5s, 20mM sodium borate buffer (pH 9.0), constant voltage of 15kV, constant capillary temperature of 25°C, and detection at 260nm. For precise measurements, uridine was used as an internal standard during optimization of the CE methods. The limits of detection and quantification for p-nitrophenyl acetate (p-NPA) were 3.01 and 9.12μM, respectively, whereas for p-nitrophenolate they were 2.05 and 6.22μM, respectively. The performance of the developed method was confirmed by determination of kinetic parameters (i.e., Km and Vmax of bCA for p-NPA); the inhibition constant (Ki) was determined for furosemide, a standard inhibitor of CA. The new method proved to be fast and efficient, and it can be used for the investigation of inhibitors of all isoforms of CAs.