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

Abstract

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.

Indirect detection of 5-hydroxytryptamine and tyramine by using tris(2,2’-bipyridyl)ruthenium-graphene modified electrode coupled with capillary electrophoresis

“…The supernatant collected in a 1.5 mL micro centrifuge tube was detected immediately. These samples were diluted to 5-fold with 10 mM pH = 7.4 PBS, and then filtrated through a 0.22 μm cellulose acetate membrane before CE analysis [25,26].…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…However, fluorescence detection requires labeling [39,41]. Conductivity [19], electrochemiluminescence [51] and mass spectrometry [16,24] offer alternative detection techniques for analytes that exhibit poor absorbance, and labeling is not practical. Mass spectrometry is especially useful when more than one product is possible and more structural information can be used to elucidate which product is being generated [16].…”

Recent developments in capillary and microchip electroseparations of peptides (2013–middle 2015)

“…An online sheath‐flow surface‐enhanced Raman scattering (SERS) detection has been applied to detection and identification of CZE separated eight biopeptides (angiotensins and other peptide hormones) with LODs at 10 nM level . CE with electrochemiluminescence detection was used to evaluate the activity of leucine aminopeptidase by monitoring cleavage of dipeptide Leu‐Pro and to determine GSH in individual Ramos cells . Indirect electrochemical detection with serial dual‐electrode detector based on electrogeneration of bromine on the first electrode and on the detection of remaining bromine (nonconsumed by the reaction with analytes in the on‐line end‐column flow‐through cell) by the downstream electrode has been applied to analysis of GSH and GSH disulfide (GSSG) in commercial GSH supplement with LODs 0.16 μM for GSH and 0.14 μM for GSSG .…”