title: Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity.
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.
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA.
authors.forename: Michal
authors.initials: M
authors.lastname: Tokmina-Roszyk
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA.
authors.forename: Dorota
authors.initials: D
authors.lastname: Tokmina-Roszyk
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA.
authors.forename: Manishabrata
authors.initials: M
authors.lastname: Bhowmick
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA. Electronic address: gfields@tpims.org.
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D047009:Hathewaya histolytica / Q000201:enzymology*; D017364:Collagenases / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D007515:Islets of Langerhans / Q000166:cytology
chemical_list: D017364:Collagenases
keywords: Bacterial collagenase; Clostripain; Collagen; FRET protease assay; Islet isolation; Stem cell isolation