A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation.
S-Adenosyl-L-methionine (AdoMet) which 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 to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) 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 microM (S,S)-AdoMet.
Cannon
Lisa M
LM
Department of Chemistry and School of Molecular Biosciences, Washington State University, Pullman 99164, USA.
Butler
Felice N
FN
Wan
Wei
W
Zhou
Zhaohui Sunny
ZS
D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D010957:Plasmids; D011107:Polylysine / Q000031:analogs & derivatives*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000032:analysis*; D013237:Stereoisomerism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014301:Trinitrobenzenes / Q000494:pharmacology
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014301:Trinitrobenzenes; C055580:trinitrobenzoylated polylysine; D011107:Polylysine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C022745:thiopurine methyltransferase