Researcher(s)
- Glorianna Yin, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Ulla-Angela Temann, Department of Medical and Molecular Sciences, University of Delaware
- Jazzlyn Jones, Department of Medical and Molecular Sciences, University of Delaware
- Esther Biswas-Fiss, Department of Medical and Molecular Sciences, University of Delaware
Abstract
Introduction: ATPases are enzymes that hydrolyze ATP into ADP, releasing energy that powers cell chemical reactions. Our laboratory studies the retina-specific ABC transporter, ABCA4. This enzyme uses ATP hydrolysis to translocate toxic byproducts of phototransduction. Some inherited genetic blinding disorders, like Stargardt, are associated with ABCA4 mutations. These mutations often affect ATP hydrolysis, which can affect transport. With nearly 4,000 variants identified in patients, it is key to understand the impact of these variants on ABCA4-ATPase. Conventionally, ATPase assays measure ATPase efficacy and function using radioisotopes like 32P. As increasing expense and stringent safety regulations cause radiometric assay usage to decline, finding effective non-radioactive alternatives is imperative.
Method: In this study, we tested BellBrook Labs’ fluorescence-based Transcreener® ADP2 FI Assay as a high-throughput, cost-effective replacement for radiometric assays. Following the manufacturer’s instructions for the assay, we used E. coli DnaB helicase as a control enzyme to test assay functionality, ease of use, and linear results. In the assay, a newly hydrolyzed ADP replaces an ADP Alexa Fluor 594 tracer bound to a quencher-conjugated ADP antibody. Each assay is a DnaB protein titration performed in triplicates in a 96-well plate along with a corresponding ATP/ADP standard curve. The samples were analyzed for fluorescence intensity with a ClarioStar plate reader.
Results: We observed linearity for DnaB concentrations of 0 ng to 2.4 ng that could be interpolated using the ATP/ADP standard curve to determine ADP percent conversion.
Conclusion: As the data displayed linearity, this assay is effective in measuring enzyme activity. It shows promise as a replacement assay for future ABCA4 ATPase experiments. With a longer shelf-life, this assay is more cost-effective in the long run than radiometric assays. The assay should be easily reproducible and applicable to other enzymes using the optimized DnaB methods.