A novel fluorometric assay for quantitative analysis of dihydrofolate reductase activity in biological samples

In an effort to study the level of dihydrofolate reductase (DHFR), the main molecular target of antifolate drugs, in healthy and malignant tissues of human origin, a new and convenient fluorometric enzymatic assay has been developed. The technique measures the overall decrease in fluorescence emission at 454 nm (lambda ex = 342 nm) due to the contributions from coenzyme oxidation and substrate reduction. This technique was developed by using an enzyme purified from beef liver. All criteria of quality were checked: sensitivity, reproducibility and specificity made it suitable for low activity measurements. It was successfully applied to human tissue crude extracts.