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.
KeywordsATPase Activity Lactate Dehydrogenase Pyruvate Kinase NADH Oxidation Coupling Enzyme
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