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

Sheldon P. Rothenberg, M. Perwaiz Iqbal, Maria Da Costa

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13 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)152-156
Number of pages5
JournalAnalytical Biochemistry
Issue number1
Publication statusPublished - 15 Mar 1980
Externally publishedYes


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