Michaelis-Menten analysis of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated microanalysis in a partiallyfilled capillary

A method for determining bovine plasma amine oxidase (PAO; EC 1.4.3.6) acti vity with benzylamine (Bz) as substrate is described. Electrophoretically m ediated microanalysis (EMMA) combined with micellar electrokinetic capillar y chromatography (MEKC) was used to perform an on-capillary enzymatic react ion and to separate the generated benzaldehyde from the other reaction prod ucts. The capillary was only partially filled with the separation solution, since the enzyme was unstable in the presence of the applied surfactant. T he initial reaction velocity of the enzyme-catalyzed reaction was estimated from the peak area of the enzyme product, benzaldehyde. An amplification s tep was introduced by means of an on-capillary incubation of 15 min, in ord er to accumulate enough reaction product to detect spectrophotometrically a t 254 nm. This setup resulted in a fully automated assay, which can be carr ied out in less then 35 min. Using the Lineweaver-Burk equation, an average Michaelis constant (K-M) for PAO was calculated to be 0.74 mM +/- 0.05 mM, which is consistent with previously reported values.