Methylation of key biological molecules and proteins plays important roles in numerous biological systems, including signal transduction, biosynthesis, protein repair, gene silencing, and chromatin regulation (1). The S-adenosylmethionine (SAM) dependent methyltransferases use SAM, the second most commonly used enzymatic cofactor after ATP. SAM, also known as AdoMet, acts as a donor of a methyl group that is required for the modification of proteins and DNA (2). Aberrant levels of SAM have been linked to many abnormalities, including Alzheimer’s Disease, depression, Parkinson’s Disease, Multiple Sclerosis, liver failure, and cancer (1,2).
Colorimetric SAM Methyltransferase BioAssay™ is a continuous enzyme-coupled assay that can continuously monitor SAM-dependent methyltransferases (3). Figure 1 outlines the general scheme of the assay. The removal of the methyl group from SAM generates S-adenosylhomocysteine (AdoHcy), which is rapidly converted to S-ribosylhomocysteine and adenine by AdoHcy nucleosidase. This rapid conversion prevents the buildup of AdoHcy and its feedback inhibition on the methylation reaction. Finally, the adenine is converted to hypoxanthine, by adenine deaminase, which in turn is converted to urate and hydrogen peroxide (H2O2). The rate of production of H2O2 is measured with the colorimetric reagent, 3,5-dichloro-2-hydroxybenzenesulfonic acid, by an increase in absorbance at 500-520 nm. The assay is supplied with AdoHcy as a positive control. The assay can be used with any purified SAM-dependent methyltransferase.
Assay Range:The detection range of this assay is from 0.013-0.133umol/min/ml of methyltransferase activity which is equivalent to an absorbance increase of 0.01 to 0.1 per minute.
Kit Components:S0070-84A: SAM Methyltransfrase Assay Buffer, 1x20mlS0070-84B: Adenosylhomocysteine, Positive Control 1mM, 1x200ul**S0070-84C: Methyltransferase Enzyme Mix, 5x300ulS0070-84D: SAM Methyltransferase Assay Buffer Additive, 1x200ulS0070-84E: SAM Colorimetric Mix, 1x1 vialS0070-84F: S-Adenosylmethionine, 3x1vial*S0070-84G: Microtiter plate, 1x96 wells**S0070-84H: HCl Assay reagent (20mM) 1x1ml
Storage and Stability:Store *S0070-84G at RT. Store **S0070-84C and **S0070-84H at -20°C. Store other components at -70°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
