Departmental Affiliation and City, State, Zip for All Authors

Alabama State University, Montgomery, AL, USA ; Department of Microbiology, Immunology and Genetics, Univerisity of North Texas Health Science Center, Fort Worth, TX, USA

Scientific Abstract

Purpose: Matrix Metalloproteinases (MMP’s) are a group of zinc dependent peptidases which can be classified based on structural differences. Out of the 26 MMP’s that have been identified, MMP-9 is of particular interest in many biomedical applications. MMP-9, also known as Gelatinase-B or 92 kDa gelatinese, is highly involved in degrading the basement membrane of the extracellular matrix (ECM). Levels of MMP-9 have been found to be upregulated in several types of cancer, including breast, bladder, ovarian, colon etc. and are generally associated with poor prognosis or prediction. The degradation activity of MMP-9 allows for tumor growth. Therefore, the overall goal of this project is to develop applications for detecting MMP-9 enzyme levels. This would result in rapid non-invasive detection, and possibly early treatment for several cancers. Approach: We can use Fluorescence resonance energy transfer (FRET) to come up with a custom peptide that is cleaved by MMP-9 enzyme, leading to easy detection and diagnosis. FRET is a distance-dependent interaction between two dye molecules in which excitation is non-radiantly transferred from a donor molecule to an acceptor molecule. This energy transfer mechanism results in the quenching (decrease in intensity) of the donor fluorescence and increase of the acceptor emission. The fluorophore with emission at the shorter wavelength acts as the donor, and instead of emitting florescence, transfers its energy to an acceptor molecule, whose emission is generally at a longer wavelength. It is a very sensitive technique that can be used for the detection of various biomolecular interactions, in particular various enzymatic reactions. FRET can also be used as a precise measurement and detection tool. Materials and Methods: We were able to successfully demonstrate FRET with a custom peptide whose partial sequence was recognized and selectively cleaved by MMP-9 enzyme. The probe uses 5/6 TAMRA (Donor) and Cy5 (Acceptor). The target peptide sequence for selected dyes is as follow: Lys-Gly-Pro-ArgSer-Leu-Ser-Gly-Lys-NH2. The fluorophores were attached to the peptide at the Ser-Leu bond, labeled on the ε-NH2 groups of lysine with donor (5/6 TAMRA) and acceptor (Cy5) dye. Peptide labelled with 5/6-TAMRA only was used as the donor control. The concentration of all fluorophores was equimolar (1 µM). Furthermore, the probe was dissolved in HBSS w/o Ca2+, Mg2+ buffer and upon addition of 2.5nM of MMP-9 enzyme, showed a gradual decrease in energy transfer over time. These measurements were done by using a 1cmx1cm quartz cuvette set-up with 530nm as the excitation wavelength. The samples were incubated at 370C and measured in 20 minute intervals.

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PHOTOPHYSICAL CHARACTERIZATION OF OLIGOPEPTIDE LINKED FRET SYSTEM IN BUFFER AND SURFACE TO DETECT LEVELS OF MATRIX METALLOPROTEINASE-9

Purpose: Matrix Metalloproteinases (MMP’s) are a group of zinc dependent peptidases which can be classified based on structural differences. Out of the 26 MMP’s that have been identified, MMP-9 is of particular interest in many biomedical applications. MMP-9, also known as Gelatinase-B or 92 kDa gelatinese, is highly involved in degrading the basement membrane of the extracellular matrix (ECM). Levels of MMP-9 have been found to be upregulated in several types of cancer, including breast, bladder, ovarian, colon etc. and are generally associated with poor prognosis or prediction. The degradation activity of MMP-9 allows for tumor growth. Therefore, the overall goal of this project is to develop applications for detecting MMP-9 enzyme levels. This would result in rapid non-invasive detection, and possibly early treatment for several cancers. Approach: We can use Fluorescence resonance energy transfer (FRET) to come up with a custom peptide that is cleaved by MMP-9 enzyme, leading to easy detection and diagnosis. FRET is a distance-dependent interaction between two dye molecules in which excitation is non-radiantly transferred from a donor molecule to an acceptor molecule. This energy transfer mechanism results in the quenching (decrease in intensity) of the donor fluorescence and increase of the acceptor emission. The fluorophore with emission at the shorter wavelength acts as the donor, and instead of emitting florescence, transfers its energy to an acceptor molecule, whose emission is generally at a longer wavelength. It is a very sensitive technique that can be used for the detection of various biomolecular interactions, in particular various enzymatic reactions. FRET can also be used as a precise measurement and detection tool. Materials and Methods: We were able to successfully demonstrate FRET with a custom peptide whose partial sequence was recognized and selectively cleaved by MMP-9 enzyme. The probe uses 5/6 TAMRA (Donor) and Cy5 (Acceptor). The target peptide sequence for selected dyes is as follow: Lys-Gly-Pro-ArgSer-Leu-Ser-Gly-Lys-NH2. The fluorophores were attached to the peptide at the Ser-Leu bond, labeled on the ε-NH2 groups of lysine with donor (5/6 TAMRA) and acceptor (Cy5) dye. Peptide labelled with 5/6-TAMRA only was used as the donor control. The concentration of all fluorophores was equimolar (1 µM). Furthermore, the probe was dissolved in HBSS w/o Ca2+, Mg2+ buffer and upon addition of 2.5nM of MMP-9 enzyme, showed a gradual decrease in energy transfer over time. These measurements were done by using a 1cmx1cm quartz cuvette set-up with 530nm as the excitation wavelength. The samples were incubated at 370C and measured in 20 minute intervals.

Manuscript Number

1035