Photophysical characterization of oligopeptide linked FRET system in PVA matrix and buffer to detect levels of matrix metalloprotease-9.

Purpose: Matrix metalloproteinases (MMP’s) are a group of zinc dependent peptidases which can be classified based on their structural differences. So far, over 26 MMP’s have been identified. Out of these, MMP-9 is of particular interest in many biomedical applications. MMP-9, also known as gelatinase B, plays an important role 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, colon, ovarian etc. and are generally associated with poor prognosis. The basement degradation activity of MMP-9 allows for tumor growth. Thus, 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 Forster 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 well-known phenomenon being used today in studying molecular interactions. Briefly; FRET is the energy transfer between two fluorophores when they are within 1-10nms of each other. 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 which can 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 and HiLyte 647 as a donor and acceptor respectively. The target peptide sequence is Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, and was optimized by Kridel et al. 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 (HiLyte647) dye. Peptide labelled with 5,6-TAMRA only was used as the donor control, and free HiLyte 647 was used as the control for acceptor. The probe and donor control were dissolved in 10% (w/w) poly-vinyl alcohol, and dried on glass slides. This produced films 200 microns in thickness. Furthermore, the probe was dissolved in buffer and upon addition of MMP-9 enzyme, showed a gradual decrease in energy transfer over time. These measurements were done by using a 1cmx1cm quartz cuvette and a square geometry set-up with 470nm as the excitation wavelength. Results: Absorption spectra and other steady state measurements indicate successful energy transfer between donor and acceptor fluorophores which gradually reduces over time as it gets cleaved by MMP-9. This showed that the peptide is functional, and also being recognized and cleaved by MMP-9 enzyme. Conclusions: It is possible to synthesize a functional FRET probe that is selectively cleaved by the enzyme MMP-9, which shows elevated levels in several cancers due to its role in basement degradation. We successfully demonstrated using FRET as a precise technique to detect and measure MMP-9 enzyme activity.