Real-Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports.
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.
Deere
Joseph
J
Department of Pure and Applied Chemistry, Thomas Graham Building, 295 Cathedral Street, University of Strathclyde, Glasgow, G1 1XL, U.K.
McConnell
Gail
G
Lalaouni
Antonia
A
Maltman
Beatrice A
BA
Flitsch
Sabine L
SL
Halling
Peter J
PJ