Study on alanine aminotransferase kinetics by microchip electrophoresis.
Alanine aminotransferase (ALT), which catalyzes the reversible conversion between L-glutamic acid (L-Glu) and L-alanine (L-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (L-Glu and L-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of L-Glu and L-Ala was conducted and limits of detection (signal/noise=3) for L-Glu and L-Ala were 4.0 × 10⁻⁷ and 2.0 × 10⁻⁷ M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (L-Ala and L-Glu), and the K(m) and V(max) values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.