DIRECT CONTINUOUS FLUOROMETRIC ASSAY FOR MONOAMINE-OXIDASE-B

The first direct and continuous fluorometric assay for monoamine oxida se B (MAO B) has been developed. E-2,5-Dimethoxycinnamylamine hydrochl oride was designed and synthesized and was found to be an excellent su bstrate for MAO B (K-m = 218 mu M, K-cat = 435 min(-1)). This compound has an intense purple fluorescence when irradiated at lambda(ex) = 34 3 nm (lambda(em) = 393 nm) in Tris buffer, pH 9.0, or sodium phosphate buffer, pH 7.2, but under the same conditions, the corresponding alde hyde, the product of the MAO-catalyzed oxidation of E-,5-dimethoxycinn amylamine hydrochloride, does not fluoresce. The activity of MAO B, th erefore, can be determined efficiently and rapidly by continuously fol lowing the decrease in fluorescence at 393 nm at enzyme concentrations as low as 100 nM. The change in fluorescence is linear up to a substr ate concentration of 500 mu M. (C) 1996 Academic Press, Inc.