Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas.

A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin-proline was used as an ECL coreagent to enhance Ru(bpy)3 (2+) ECL signals efficiently. The detection limit for proline was 2.88 × 10(-6) m (signal-to-noise ratio 3), which was equal to 9.60 × 10(-8) units of LAP being used to catalyze leucin-proline for 1 min. The Michaelis constant Km (2.07 × 10(-2) mol/L) and the maximum reaction velocity Vmax (1.06 × 10(-5) mol/L/min) of LAP for leucin-proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method.