Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II

This chapter describes the use of a common spectrophotometric assay for following the rate of ATP hydrolysis as applied to type II DNA topoisomerases. It is called a “coupled assay,” because each time an ATP molecule is hydro-lyzed, a molecule of NADH is rapidly oxidized; ATP hydrolysis and NADH oxidation are therefore coupled. Although NADH absorbs strongly at 340 nm, the product of its oxidation, NAD⁺ , does not. Therefore, the rate of ATP hydrolysis can be determined by following the decrease in optical absorbance of the reaction at 340 nm.