Use of 10-formyl-5,8-dideazafolate (cas 61038-31-1) as substrate for rat 10-formyltetrahydrofolate dehydrogenase

Add time:07/11/2019    Source:sciencedirect.com

Publisher SummaryThis chapter discusses the use of 10-formyl-5,8-dideazafolate (cas 61038-31-1) as substrate for rat 10-formyltetrahydrofolate dehydrogenase. 10-formyltetrahydrofolate (10-HCO-H4PteGlu) is susceptible to oxidative degradation and must be protected by reducing agents during synthesis and use in enzyme assays. The (6R,S)-10-HCO-H4PteGlu, while highly unstable, is easily generated from stable, commercially available (6R,S)-5-HCO-HaPteGlu in the presence of 2-mercaptoethanol (2-ME), using the method of Rabinowitz. The 10-formyl-5,8-dideazafolate was originally synthesized as a quinazoline analog of folic acid. It was found to be a modest inhibitor of rat liver dihydrofolate reductase and had activity against L1210 leukemia in mice. The oxidation of 10-formyl-5,8-dideazafolate is followed by the production of 5,8-dideazafolate at 295 nm 6 or NADPH at 340 nm. The absorption of 5,8-dideazafolate at 340 nm is reflected in the adjusted extinction coefficient for NADPH. The absorption of NADPH at 295 nm is approximately 2% of the extinction coefficient for 5,8-dideazafolate and is ignored. Hydrolase activity is followed by the production of 5,8-dideazafolate at 295 nm in the presence of 100 mM 2-ME. The assay may be run at room temperature. Assays of crude extracts require the removal of low molecular weight compounds by spin column desalting.

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