Methodology Development of the Conversion of 3-Methylxanthine to Theobromine by TcCS2 and Xanthine to 3-Methylxanthine by PcCS1 Using ESI-MS
Lee, Ryan A.
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Proteins, more specifically enzymes, are responsible for day to day functions of all organisms and for this reason they are studied by biologists and chemists alike to understand them. The fields of chemistry and biology share many common interests, one of them being these enzymes. Current methodologies for studying enzymes may require a spectral component in UV-Vis spectroscopy or fluorescence. These methodologies may not work for all compounds, for this reason this methodology using ESI-MS is being developed. On top of catering to many compounds it is also fast and very reproducible. Analysis was done by taking reaction mixture containing the converted products theobromine and 3-methylxanthine and their respective enzymes TcCS2 and PcCS1 and injecting them directly into the ESI-MS. Using this methodology, it was determined that the detection of the converted products was feasible but at this stage quantification was not. The first problem to overcome was the salt concentration. Sodium is sometimes necessary for the preparation of the enzyme environment and as a result having a high sodium concentration can create sodium adducts making quantification difficult.2 The second problem was suspected to be ion suppression; it was hypothesized that something in the reaction mixture was suppressing the charge of the products. This problem requires further insight to solve, but when it is solved quantification using this methodology could be possible.