Assay for catechol-O-methyltransferase in erythrocytes using a new fluorogenic substrate, 2-(3,4-dihydroxyphenyl)naphtho[1,2-d]thiazole.
A highly sensitive method for the assay of catechol-O-methyltransferase in erythrocytes is described, which employs high-performance liquid chromatography with fluorescence detection. A newly synthesized catechol compound, 2-(3,4-dihydroxyphenyl)naphtho [1,2-d]thiazole is used as a highly fluorogenic substrate for catechol-O-methyltransferase; the m- and p-methylated products formed enzymatically from the substrate under the optimum conditions, after extraction with n-hexane--chloroform, are separated by normal-phase chromatography on LiChrosorb Si 100. The limits of detection for m- and p-methylated products are 3 pmol per assay tube (60 fmol per injection volume of 20 microliter) in each case. The ratio of m- and p-methylated products was 0.54. This method requires as little as 50 microliter of human erythrocytes.
D052160:Benzothiazoles; D002394:Catechol O-Methyltransferase / Q000097:blood*; D065098:Catechol O-Methyltransferase Inhibitors; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008745:Methylation; D011748:Pyrogallol / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013844:Thiazoles
D052160:Benzothiazoles; D065098:Catechol O-Methyltransferase Inhibitors; D005456:Fluorescent Dyes; D013844:Thiazoles; D011748:Pyrogallol; C042353:2-(3,4-dihydroxyphenyl)naphtho(1,2-d)thiazole; D002394:Catechol O-Methyltransferase