A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases.
A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect S-adenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 microM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 muM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-O-methyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proof-of-principle experiments. A time- and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC(50) value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity.
Graves
Tiffany L
TL
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
Zhang
Yi
Y
Scott
John E
JE
D001577:Benzophenones / Q000494:pharmacology; D002394:Catechol O-Methyltransferase / Q000378:metabolism; D065098:Catechol O-Methyltransferase Inhibitors; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D008780:Methyltransferases / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000032:analysis; D012436:S-Adenosylmethionine / Q000032:analysis*
D001577:Benzophenones; D065098:Catechol O-Methyltransferase Inhibitors; C071721:Ro 41-0960; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; D002394:Catechol O-Methyltransferase