Enzymatic Treatment to Eliminate the Extracellular ATP for Improving the Detectability of Bacterial Intracellular ATP

A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin–luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP, which are not active to the L-L system. In the model system (3.2 × 10⁻⁸mATP in 1% yeast extract solution) the treatment with adenosine phosphate deaminase resulted in the reduction of ATP to 1.3 × 10⁻¹¹m, and the concomitant use of apyrase lowered the concentration to 3.3 × 10⁻¹³m. The treatment (0.05 U/ml of adenosine phosphate deaminase and apyrase) was applied to the detection of bacteria in broth by the L-L method, affording the detection of 42 colony-forming unit (CFU)/ml ofEscherichia coliand 10 CFU/ml ofStaphylococcus aureusin the broth.