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

P.J. Halling; J. Deere; B.A. Maltman; S.L. Flitsch; G. McConnell; A. Lalaouni

doi:10.1002/adsc.200700044

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

P.J. Halling, J. Deere, B.A. Maltman, S.L. Flitsch, G. McConnell, A. Lalaouni

Research output: Contribution to journal › Article

8 Citations (Scopus)

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.

Original language	English
Pages (from-to)	1321-1326
Number of pages	6
Journal	Advanced Synthesis and Catalysis
Volume	349
Issue number	8-9
DOIs	https://doi.org/10.1002/adsc.200700044
Publication status	Published - 2007

Keywords

protease cleavage
two-photon microscopy
spatial resolution
solid-phase biocatalysis
real-time imaging

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10.1002/adsc.200700044

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title = "Real time imaging of protease action on substrates covalently immobilised to polymer supports",

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.",

keywords = "protease cleavage, two-photon microscopy , spatial resolution , solid-phase biocatalysis, real-time imaging",

author = "P.J. Halling and J. Deere and B.A. Maltman and S.L. Flitsch and G. McConnell and A. Lalaouni",

year = "2007",

doi = "10.1002/adsc.200700044",

language = "English",

volume = "349",

pages = "1321--1326",

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issn = "1615-4150",

number = "8-9",

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TY - JOUR

T1 - Real time imaging of protease action on substrates covalently immobilised to polymer supports

AU - Halling, P.J.

AU - Deere, J.

AU - Maltman, B.A.

AU - Flitsch, S.L.

AU - McConnell, G.

AU - Lalaouni, A.

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

KW - protease cleavage

KW - two-photon microscopy

KW - spatial resolution

KW - solid-phase biocatalysis

KW - real-time imaging

U2 - 10.1002/adsc.200700044

DO - 10.1002/adsc.200700044

M3 - Article

VL - 349

SP - 1321

EP - 1326

JO - Advanced Synthesis and Catalysis

JF - Advanced Synthesis and Catalysis

SN - 1615-4150

IS - 8-9

ER -