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Real‐Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports
Author(s) -
Deere Joseph,
McConnell Gail,
Lalaouni Antonia,
Maltman Beatrice A.,
Flitsch Sabine L.,
Halling Peter J.
Publication year - 2007
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200700044
Subject(s) - subtilisin , chemistry , covalent bond , cleavage (geology) , fluorescence , ethylene glycol , trypsin , polymer , protease , fluorescence microscope , polymer chemistry , organic chemistry , enzyme , physics , geotechnical engineering , quantum mechanics , fracture (geology) , engineering
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.