NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities.

Genome database searches retrieve over 10,000 sequences that either are known to use NTP or contain motifs suggestive of that. Hence, ATP hydrolysis is a convenient tool for monitoring their biological activity. Among the most popular assays are measurements of the release of Pi, ADP, or Hþ [1]. For enzymes sensitive to the accumulation of ADP, an assay based on ADP recycling coupled to the oxidation of NADH represents a particular advantage [2,3]. The high molar absorption coefficient of NADH and the virtual absence of protein or DNA absorbance at this wavelength ensure reliable ATP hydrolysis measurements down to the micromolar concentration scale. A limitation of this technique is that it requires investment into relatively costly equipment with a limited capacity for parallel analysis. Microplate readers are devoid of these limitations, offering parallel analysis of many samples and great versatility. However, the lightpath of a microplate reader is variable and depends on the well fill volume, requiring proper calibration. Herein, we adapted the NADH-coupled ATPase assay for a 96-well microplate reader. A previous adaptation of this assay to the microplate format focused on P-glycoprotein, one particular ATPase [4]. Here, we elaborated general assay parameters to analyze a variety of ATPases with low and high specific activities. We show that beyond the numerous advantages of microplate readers (high throughput, low cost, ease of operation, use of disposable plasticware in place of quartz cells), the microplates ensure sufficiently high sensitivity and precision to measure ATP hydrolysis down to the submicromolar scale. We recommend practical tips for adapting the microplate-based assay for kinetic studies