Elsevier

Analytical Biochemistry

Volume 308, Issue 2, 15 September 2002, Pages 358-363
Analytical Biochemistry

A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

https://doi.org/10.1016/S0003-2697(02)00267-1Get rights and content

Abstract

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 60μM (S,S)-AdoMet.

Section snippets

Materials

AdoMet p-toluenesulfonate salt and ATP disodium salt were purchased from Sigma. 5,5-Dithiobis-2-nitrobenzoic acid (DTNB; Ellman's reagent) was purchased from Aldrich. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Fluka. l-Methionine, isopropyl-β-d-thiogalactoside (IPTG), dithiothreitol (DTT), and kanamycin were purchased from Fisher. HiTrap Chelating HP columns were purchased from Amersham Pharmacia. All other chemicals were of the highest purity available.

Preparation of reagents

A solution of

Results and discussion

Ames and co-workers [21] reported that human thiopurine S-methyltransferase methylates a series of aromatic thiols, including 4-nitrothiophenol, using AdoMet as the methyl donor. Since methylation of 4-nitrothiophenol is associated with a significant UV–visible spectral change, we reasoned that a TPMT-catalyzed thiol methylation could be used to quantify AdoMet. From 1 L of growth medium, about 100 mg recombinant human TPMT was purified to homogeneity as judged by Coomassie-stained SDS–PAGE gel.

Acknowledgements

We are indebted to Dr. Richard Weinshilboum at Mayo Clinics for providing human TPMT cDNA, Dr. G. Doug Markham at Fox Chase Cancer Center for providing AdoMet synthetase expression strain and purified enzyme, and Drs. Kenneth A. Cornell and Michael K. Riscoe at the Portland VA Medical Center for providing AdoHcy nucleosidase expression strain and purified enzyme. We also thank Drs. Cornell and Markham for comments and suggestions; L.M.C. and F.N.B. were supported by a mini-grant from the

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