Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots‐peptide assembly

摘要

Abstract Herein, a novel assay has been developed for monitoring PreScission protease (His‐PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO‐LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE‐FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His‐tagged ATTO 590 labeled peptide substrate (ATTO‐LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the F?rster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO‐LEVH6‐QD assembly was then incubated with His‐PSP to study the proteolytic cleavage of surface bound ATTO‐LEVH6 by CE‐FL. Our data suggests that PreScission‐mediated proteolytic cleavage is enzyme concentration‐ and incubation time‐dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases.