A sterospecific colorimetric assay for (s,s)-Adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation
Cannon, Lisa M.
Butler, Felice N.
Zhou, Zhaohui Sunny
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S-Adenosyl-L-methionine (AdoMet or SAM) that is biologically synthesized by AdoMet synthetase bears an s-configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of s-and R-isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R-isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (s,s)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (s,s)-AdoMet reacts with 2-nitro-5-thiobenzoic acid (TNB) to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 220.127.116.11), and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows down the assay. AdoHcy nucleosidase (EC 18.104.22.168) irreversibly cleaves AdoHcy to adenine and s-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 f.lM (s,s)AdoMet.