Capillary electrophoresis-based nanoscale assays for monitoring ecto-5′-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Abstract

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5′-nucleotidase (ecto-5′-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5′-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 μM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10 mM Hepes [pH 7.4], 2 mM MgCl 2, and 1 mM CaCl 2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 μM i.d.), followed by enzyme (recombinant rat ecto-5′-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5′-NT.