ASSAY FOR TYROSINE HYDROXYLATION ACTIVITY OF TYROSINASE FROM BETALAIN-FORMING PLANTS AND CELL-CULTURES

In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibl y involved in betalain biosynthesis, we have evaluated different assay s for the detection and quantification of the enzymatic product Dopa w ith respect to sensitivity, simplicity, and suitability for automatiza tion. A tyrosinase assay including reversed-phase high-performance liq uid chromatography with isocratic elution and fluorescence detection h as been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm). This improved assay was sensitive (detection limi t: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 p mol-20 nmol Dopa). The method proved to be suitable for high-performan ce Liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues o f Portulaca grandiflora. (C) 1996 Academic Press, Inc.