Fluorescence-Based On-Resin Detection of Three Model Proteases

David Milićević,Jan Hlaváč

doi:10.3390/chemosensors9120359

Abstract

A new approach to on-resin detection of three model proteases (trypsin, chymotrypsin, and thrombin) has been developed, while at the same time already described methodology for simultaneous detection of two enzymes (trypsin and chymotrypsin) has been additionally generalized. Appropriate immobilized substrates, comprising specifically cleavable peptide sequences capped with fluorescent dyes, have been synthesized on Rink Amide PEGA resin or Amino PEGA resin modified with backbone amide linker (BAL). Resulting solid support-bound probes were then dispersed into Tris-HCl buffer solution (pH = 8.0) and subjected to enzymatic cleavage. Liberated fluorophores have been tracked by fluorescence measuring. The competitive activities of studied proteases towards the thrombin probe have been efficiently limited and controlled by employing a Bowman-Birk inhibitor into a system.

Highlights

IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
A concept of a cleavable peptide linkers embedded to a solid support was successfully implemented in our research group [23], as we developed on-resin sensor for simultaneous detection of trypsin and chymotrypsin in a sample
With the aim of on-resin enzyme assays, the trypsin and chymotrypsin probes were synthesized on Rink Amide PEGA resin (0.35 mmol/g, Novabiochem, Darmstadt, Germany), while the thrombin probe was prepared on Amino PEGA resin (0.42 mmol/g, Novabiochem, Darmstadt, Germany)