Conjugated Polyelectrolyte Based Real-Time Fluorescence Assay for Adenylate Kinase

Abstract

Addition of adenosine 5'-triphosphate (ATP) to a solution of the anionic conjugated polyelectrolyte PPECO2 and copper(II) ion (Cu2+) recovers the Cu2+-quenched fluorescence of PPECO(2) to a significantly greater extent compared with the addition of adenosine 5'-diphosphate (ADP) or adenosine 5'-monophosphate (AMP) at the same concentration levels. Taking advantage of the differential response of the PPECO2-Cu2+ system to ATP, ADP and AMP, we have developed fluorescence turn-off and turn-on assays that monitor the catalytic activity of adenylate kinase (ADK) in the equilibrium transphosphorylation reaction (ATP + AMP <--> 2ADP). The fluorescence turn-on and turn-off assays monitor the forward and reverse transphosphorylation reactions, respectively. The forward assay operates with ATP substrate present at the submillimolar concentration range and offers a straightforward and rapid detection of ADK catalytic activity with the enzyme present in the nanomolar range, in either end-point or real-time formats. The real-time fluorescence intensity from PPECO2 can be converted to substrate (ATP) concentration in the forward reaction assay by using an ex-situ calibration curve, allowing ADK catalyzed reaction rates and kinetic parameters to be determined. ADK activation by Mg2+ and inhibition by Ag+ and product are analyzed using the optimized assay system. Non-specific interactions are observed between the assay complex and other proteins, but the signal response to the ADK assay is demonstrated to mainly arise from the specific enzyme catalyzed transphosphorylation reaction.