- Improved assay platform with BMG LABTECH readers and CyDye™ labelled peptides
- Enhanced and significant fluorescence increase compared to traditional fluors
- Development of CyDye™ labelled substrates to control MMP activity
Table of contents
Members of the matrix metalloproteinase (MMP) family play an important role in tissue remodeling and repair. However, inappropriate MMP activity has been implicated in a number of disease states including arthritis, tumour invasion and metastasis, and cardiovascular disease. The development of agents to control MMP activity continues to be a major focus for the pharmaceutical industry.
Many synthetic MMP peptide substrates have been described which incorporate a fluorophore and a quencher moiety positioned on either side of the enzyme cleavage site. We have prepared a number of CyDyeTM labelled MMP substrates. Cleavage of these substrates with specific MMP enzymes produces improved and significant fluorescence increase at donor fluorophore wavelengths (Figure 1), when compared to traditionally used fluors.
The POLARstar® Omega and PHERAstar® FS have 2 Photo Multiplier Tubes (PMT) and hence do not have to switch filters or polarizers to read the second channel, Simultaneous Dual Emission (SDE) makes assays like FRET faster and more precise.
Material & Experimental
Peptides were synthesized and labelled with active CyDye esters (Amersham Biosciences) using standard synthesis and labelling procedures. Following purification by RP-HPLC and confirmation of purity by mass spectrophotometry, dual labelled peptide was freeze-dried. Prior to use, labelled peptide substrates (Table 1) were dissolved in DMSO and stocks were stored at -20°C. Peptide Mca-PLGL-Dpa-AR-NH2, supplied as a freeze-dried powder (CN Biosciences), was dissolved in methanol as per manufacturer’s instructions.
Table 1: Peptide sequences for cleavage assay
|Peptide I||Cy3B-PLG↑ LAARK-Cy5Q|
|Peptide II||Cy3B-PLG↑ LFARK-Cy5Q|
|Peptide III||Mca-PLG↑ L-Dpa-AR|
Dpa, 3-(2,4-Dinitrophenyl)-L-2,3-diaminopropionic acid
Activation of MMP-2:
Human recombinant MMP-2 pro-enzyme (CN Bio-sciences) was activated by incubation in assay buffer containing 4-aminophenyl mercuric acetate for 2 hours at 37°C.
Labelled peptides (400 nM) were incubated at 37°C with or without activated MMP-2 enzyme in assay buffer (50 mM Tris pH 7.5, containing 150 mM NaCl, 10 mM CaCl2, 10 μM ZnCl2, 0.05% (w/v) BrijTM-35 and 0.05% NaN3). Assays were configured in black, opaque 384-well plates (Greiner) in final reaction volumes of 60 μL. Plates were read on BMG LABTECH fluorescence plate reader using 320/390 nm and 530/570 nm excitation and emission wavelengths for Mca and CyTM3B respectively.
Results & Discussion
We have prepared two MMP substrates that contain Cy3B and Cy5Q as a fluorophore/quencher pair. In a series of experiments, these were compared with each other and with the well characterized peptide Mca-PLGL-Dpa-AR. A time course for hydrolysis of the substrates by MMP-2 was established (Figure 2) and all three peptides were found to be effectively cleaved.
At the assay endpoint we calculated signal/background (S/B) values for the three peptides and the recognized Z’ statistical factor for peptides I and II (Table 2). Overall, results showed that peptide II was the more favourable substrate for MMP-2.
Table 2: Summary statistics for MMP-2 assay.
We have employed CyDye fluors in a FRET protease cleavage assay for the enzyme MMP-2. Substrates combining the fluorescent donor (Cy3B) with a Cy5Q quencher in a de-quench assay format were compared with an equivalent substrate incorporating a methyl-coumarin fluor and dinitrophenyl based quencher.
All of the peptide substrates (Figure 2) were efficiently hydrolyzed by the MMP-2 enzyme. Signal increases, measured on the BMG LABTECH fluorescence microplate reader, were >25-fold following hydrolysis of the CyDye labelled substrates, compared with only a 9-fold signal increase following hydrolysis of the Mca/Dpa labelled substrate (when evaluated by time course analysis). For both of the CyDye labelled peptides, no significant signal increases were observed in control (no enzyme containing) wells. This combination of CyDye labelled peptides and detection on the BMG LABTECH reader provides an improved assay platform when compared with more traditionally used fluors (such as Mca).
The two CyDye peptides differ at the P2’ position (Table 1). Other well characterized substrates contain either tryptophan or Dpa at this site. The data presented here suggest that phenylalanine is also a favourable residue in subsite P2’. This is in line with the observation that MMP enzymes favour aromatic side chains at P2’.
GE, imagination at work and GE monogram are trademarks of General Electric Company
CyDye and Cy are trademarks of GE Healthcare companies
CyDye: This product or portions thereof is manufactured under an exclusive licence from Carnegie Mellon University under US patent number 5,268,486 and equivalent patents and patent applications in other countries.
Cy3B: This product is manufactured under an exclusive licence from Carnegie Mellon University and is covered by US patent number 6, 133,445 and equivalent patents and patent applications in other countries.
CyQ (or Cy5Q or Cy7Q): These products are covered under US patent number 6,828,116 and equivalent patents and patent applications in other countries in the name of GE Healthcare UK Limited.
The purchase of CyDye products includes a limited licence to use the CyDye products for internal research and development, but not for any commercial purposes. A licence to use the CyDye products for commercial purposes is subject to a separate licence agreement with GE Healthcare.
All third party trademarks are the property of their respective owners. ©2007 General Electric Company - All rights reserved.
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84 Uppsala, Sweden.
GE Healthcare Europe GmBH, Munzinger Strasse 5, D-79111 Freiburg, Germany.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK.
GE Healthcare Bio-Sciences Corp., 800 Centennial Avenue, P.O.Box 1327, Piscataway, NJ 08855-1327, USA.
GE Healthcare Bio-Sciences KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan.