title: Fluorescence detection of MMP-9. II. Ratiometric FRET-based sensing with dually labeled specific peptide.
abstract: In our previous paper we showed that the MMP-9 enzyme recognizes a specific peptide sequence, Lys-Gly- Pro-Arg-Ser-Leu-Ser-Gly-Lys, and cleaves the peptide into two parts [1]. In this study, the peptide is labeled with two dyes, carboxyfluorescein (5-FAM) and Cy5. A highly efficient energy transfer of over 80% results in a dominant emission of Cy5 at ~670 nm with an excitation of 470 nm. Severance of the peptide by the MMP-9 enzyme eliminates Förster Resonance Energy Transfer (FRET) and strongly increases the fluorescence of the 5-FAM dye. In this manuscript we describe the strategy for a FRET-based method for MMP-9 enzyme detection. The basic aim is to apply a ratio-metric sensing technique in which a ratio of green/red fluorescence intensity is measured as a function of enzyme concentration. The ratio-metric method eliminates many experimental variables and enables accurate MMP-9 detection.
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Ryan
authors.initials: R
authors.lastname: Rich
authors.forename: Anindita
authors.initials: A
authors.lastname: Mukerjee
authors.forename: Amalendu P
authors.initials: AP
authors.lastname: Ranjan
authors.forename: Jamboor K
authors.initials: JK
authors.lastname: Vishwanatha
authors.forename: Anna K
authors.initials: AK
authors.lastname: Kurdowska
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Julian
authors.initials: J
authors.lastname: Borejdo
authors.affiliation: Center of Commercialization of Fluorescence Technologies (CCFT), Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, TX 76107, USA. Rafal.Fudala@unthsc.edu.
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
mesh_terms: D002478:Cells, Cultured; D003116:Color; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000032:analysis*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010455:Peptides / Q000737:chemistry*; D011874:Radiometry
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D020780:Matrix Metalloproteinase 9