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Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas
Author(s) -
Su Ming,
Wei Min,
Zhou Zhixin,
Liu Songqin
Publication year - 2013
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.2890
Subject(s) - chemistry , electrochemiluminescence , capillary electrophoresis , proline , chromatography , detection limit , leucine , aminopeptidase , analytical chemistry (journal) , proline dehydrogenase , hydrolysis , michaelis–menten kinetics , amino acid , enzyme , enzyme assay , biochemistry
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 K m (2.07 × 10 −2 mol/L) and the maximum reaction velocity V max (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. Copyright © 2013 John Wiley & Sons, Ltd.