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Anal Biochem. 2014 May 15;453:61-9. doi: 10.1016/j.ab.2014.02.024. Epub 2014 Mar 6.

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity.

Author information

1
Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA.
2
Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA. Electronic address: gfields@tpims.org.

Abstract

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] had a melting temperature (Tm) of 36.2°C and was hydrolyzed efficiently by bacterial collagenase (k(cat)/K(M)=25,000s(-1)M(-1)) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.

KEYWORDS:

Bacterial collagenase; Clostripain; Collagen; FRET protease assay; Islet isolation; Stem cell isolation

PMID:
24608089
PMCID:
PMC4260936
DOI:
10.1016/j.ab.2014.02.024
[Indexed for MEDLINE]
Free PMC Article

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