A simplified radioenzymatic assay for dihydrofolate reductase using [3H]dihydrofolate

✍ Scribed by Sheldon P. Rothenberg; M. Perwaiz Iqbal; Maria Da Costa

• Publisher: Elsevier Science
• Year: 1980
• Tongue: English
• Weight: 326 KB
• Volume: 103
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(80)90249-3

✦ Synopsis

This report describes a simple method to measure the activity of dihydrofolate reductase using the substrate [3H]dihydrofolate, which is generated by preincubation of [3H]folic acid for 10 min with dithionite before the enzymatic reaction. The procedure then measures the direct reduction of [3H]dihydrofolate to [3H]tetrahydrofolate by coprecipitating the unreduced substrate with excess unlabeled folic acid and acidified zinc sulfate. The advantage of this method is that [3H]dihydrofolate, which is not commercially available, can be generated from high specific activity [3H]folic acid, which is commercially available, immediately before initiating the enzymatic reaction. By this modification, the two important advantages of radioenzymatic assays for dihydrofolate reductase can be more easily exploited; namely, increased sensitivity because much less substrate need be used, and the ability to measure enzyme activity in crude tissue preparations without interference by precipitating proteins or nucleotide oxidases.