Conjugated Polyelectrolyte Based Fluorescence Turn-On Assay for Real-Time Monitoring of Protease Activity

A fluorescence turn-on assay for monitoring protease activity is developed on the basis of a water-soluble carboxylated polyfluorene derivative, PFP-CO2Na, and its different fluorescence response toward cytochrome c (cyt c) and its fragments. PFP-CO2Na is synthesized via Suzuki coupling polymerization between 2,7-dibromo-9,9-bis(3'-tert-butyl propanoate)fluorene and 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-y1)-benzene, followed by treatment with trifluoroacetic acid and Na2CO3. The fluorescence of PFP-CO2Na can be significantly quenched by cyt c due to complexation-mediated electron transfer between the polymer and protein. Using the complex of PFP-CO2Na/cyt c as a substrate, a real-time fluorescence turn-on assay for trypsin activity study has been developed. Addition of trypsin to the substrate solution induces gradual recovery of the fluorescence intensity for PFP-CO2Na due to trypsin-catalyzed hydrolysis of cyt c, which dissociates the heme moiety from the polymer vicinity. The time-dependent fluorescence intensity increase of PFP-CO2Na in the presence of trypsin allows us to derive the initial reaction rates and K-cat/K-m (5350 M-1 s(-1)) for trypsin-catalyzed hydrolysis. Addition of trypsin inhibitor efficiently inhibits trypsin-catalyzed hydrolysis reaction of cyt c, which leads to a decreased fluorescence turn-on response of PFP-CO2Na.