High-performance liquid chromatographic assay for catechol-O-methyltransferase in human and rat erythrocyte membrane and soluble fractions, and rat tissues.
The previously reported assay method for catechol-O-methyltransferase, which uses 2-(3,4-dihydroxyphenyl)naphtho[1,2-d]thiazole as a fluorogenic substrates, was applied to the assay of the enzyme in human and rat erythrocyte membrane and soluble fractions, and various rat tissue preparations, with some modifications. The m- and p-methylated products formed enzymatically were determined by normal-phase high-performance liquid chromatography with fluorescence detection. The detection limits for the m- and p-methylated products were 1 pmol per assay tube (50 fmol per injection volume of 50 .mu.l) in each case. No difference among the preparations was observed in optimal reaction conditions for the enzyme, but the ratio of m- and p-methylated products and the Michaelis constant values for the substrate and S-adenosyl-L-methionine (methyl donor) varied from preparation to preparation.