Real-Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports

Overview

Journal Adv Synth Catal

Date 2009 Sep 26

PMID 19779571

Citations 2

Authors

Joseph Deere

Gail McConnell

Antonia Lalaouni

Beatrice A Maltman

Sabine L Flitsch

Peter J Halling

Affiliations

Soon will be listed here.

Abstract

We report for the first time single bead spatially resolved activity measurements of solid-phase biocatalytic systems followed in real-time. Trypsin cleavage of Bz-Arg-OH and subtilisin cleavage of Z-Gly-Gly-Leu-OH each liberate a free amino group on aminocoumarin covalently immobilised to PEGA(1900) beads [a co-polymer of poly(ethylene glycol) with molecular mass of 1900 cross-linked with acrylamide]. This restores fluorescence which is imaged in optical sections by two-photon microscopy. For trypsin cleavage, fluorescence is restricted initially to surface regions, with more than 1 hour needed before reaction is fully underway in the bead centre, presumably reflecting slow enzyme diffusion. In contrast, for subtilisin cleavage fluorescence develops throughout the bead more quickly.

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