The authors relied on a methylation-sensitive endoproteinase, which cleaves unmethylated peptide but not methylated peptide.49 The resultant digested peptide and undigested peptide had been resolved by microfluidic capillary electrophoresis based on their distinctive charge-to-mass ratios. With G9a as being a model PMT, the authors demonstrated the strategy is extremely quantitative and ideal for characterizing the kinetics of PMT-catalyzed reactions.49 MS- and antibody-based approaches have also been used to measure the byproduct SAH in PMT-catalyzed reactions . The Frankel lab reported a tandem MS/MS method to quantify SAH. 54 With this particular assay, they had been capable to quantify the sources creating SAH background in PRMT1-catalyzed reactions and concluded that, besides the SAH from your contamination in business SAM and from SAMs nonenzymatic decomposition , automethylation of PRMT1 accounts to get a portion on the observed SAH background.
54 The byproduct SAH in PMT-catalyzed reactions may also be quantified by antibody-based aggressive assays . Capdevila et. al. to begin with reported a competitive immunoassay making use of SAH-BSA conjugate order Triciribine and anti-SAH antibody to quantify SAH in plasma.55 In this assay, SAH competes with microplate-coated SAH-BSA to bind anti-SAH antibody and hence reduces ELISA signal from the microplate-immobilized antibody. Graves et. al. created a related competitive assay with fluorescein-SAH and anti-SAH antibody.56 In Gravess approach, SAH is quantified by competing fluorescein-SAH to bind the antibody and hence trigger the loss of fluorescence polarization signal.
The assay has demonstrated its feasibility for catechol-Omethyltransferase and is probable applicable to PMTs, given their shared byproduct SAH.56 Yet, one particular need to be cautious to make use of the SAH-based Orotic acid fluorescence polarization since the readout is linear only inside a narrow variety of SAH concentration .56 Quite a few SAH-based quantification assays have been created for small-molecule methyltransferases such as salicylic acid methyltransferase57 and catechol-Omethyltransferase. 58 The Zhou laboratory reported an enzyme-coupled chromogenic assay for salicylic acid methyltransferase.57 This assay relied on two coupling enzymes MTAN and LuxS to convert SAH into homocysteine . Homocysteine can then be quantified with Ellmans reagent . The Hrycyna laboratory reported a comparable fluorogenic assay for catechol-Omethyltransferase .
58 This assay relies on the coupling enzyme SAH hydrolase to procedure SAH into homocysteine, that’s then quantified by a free-thiol-activated dye fluorescein-cystamine-methyl red. The Trievel laboratory formulated the first SAH-based quantification assay for PMTs.