Essay
A sterospecific colorimetric assay for (s,s)-Adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation
2002
Handle:
https://hdl.handle.net/2376/2433
Abstract
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 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 down 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 f.lM (s,s)AdoMet.
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Details
- Title
- A sterospecific colorimetric assay for (s,s)-Adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation
- Creators
- Lisa M. Cannon (Author)Felice N. Butler (Author)Wei Wan (Author)Zhaohui Sunny Zhou (Author)
- Contributors
- Zhaohui Sunny Zhou (Advisor)
- Academic Unit
- Honors Theses (WSU Pullman, Passed with Distinction)
- Identifiers
- 99900590552801842
- Copyright
- http://rightsstatements.org/vocab/InC/1.0/; http://www.ndltd.org/standards/metadata; http://purl.org/eprint/accessRights/OpenAccess; In copyright; Publicly accessible; openAccess
- Language
- English
- Resource Type
- Essay