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Real time imaging of protease action on substrates covalently immobilised to polymer supports

Halling, P.J. and Deere, J. and Maltman, B.A. and Flitsch, S.L. and McConnell, G. and Lalaouni, A. (2007) Real time imaging of protease action on substrates covalently immobilised to polymer supports. Advanced Synthesis and Catalysis, 349 (8-9). pp. 1321-1326. ISSN 1615-4150

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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 PEGA1900 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.