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

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.