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-yl)-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.
Wang
Yanyan
Y
Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore.
Zhang
Yong
Y
Liu
Bin
B
D055162:Biocatalysis; D045304:Cytochromes c / Q000378:metabolism; D004573:Electrolytes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D013997:Time Factors; D014357:Trypsin / Q000032:analysis* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D004573:Electrolytes; D005456:Fluorescent Dyes; D014361:Trypsin Inhibitors; D045304:Cytochromes c; D014357:Trypsin