title: Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities.
abstract: A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.
authors.affiliation: Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA.
authors.forename: Ismail H
authors.initials: IH
authors.lastname: Al-Abdullah
authors.affiliation: Department of Molecular Immunology, Beckman Research Institute of the City of Hope, USA.
authors.forename: Karine
authors.initials: K
authors.lastname: Bagramyan
authors.affiliation: Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA.
authors.forename: Shiela
authors.initials: S
authors.lastname: Bilbao
authors.affiliation: Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute, Beckman Research Institute of the City of Hope, USA.
authors.forename: Meirigeng
authors.initials: M
authors.lastname: Qi
authors.affiliation: Department of Molecular Immunology, Beckman Research Institute of the City of Hope, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Kalkum
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010196:Pancreatic Elastase / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D017364:Collagenases; D013820:Thermolysin