SensoLyte™ 520 MMP Assay Kits
Among the 16 5-FAM/ QXL 520 FRET peptides designed for MMP screening, one peptide showed rapid cleavage by most of the MMPs tested. Using this substrate, AnaSpec developed the SensoLyte 520 Generic MMP Assay Kit for use in measuring the activity of MMP-1, 2, 3, 7, 8, 9, 10, 12, 13, and 14 (Figure 2). This is a homogeneous kit, ideal for high-throughput screening of MMP inducers and inhibitors using purified MMPs. Pro-MMPs enzymes should be activated with 1 mM 4-aminophenylmercuric acetate (APMA) immediately before the experiment and should be activated at a higher protein concentration. After activation, the enzyme can be diluted and used in the assay.
The assay is performed in a convenient 96-well microplate format and takes minimal hands-on time. Activated MMPs are diluted in assay buffer to appropriate concentrations and added to microplate wells with test compounds. The suggested total volume of MMP diluent and test compound for a 96-well plate is 50 µL. The suggested controls for the assay are positive control (MMP enzyme only), inhibitor control (enzyme and known MMP inhibitor), vehicle control (MMP diluent and vehicle used to deliver test compound), test compound control (assay buffer and test compound), and substrate control (assay buffer only).
The generic MMP substrate should be diluted 1:100 in assay buffer and added to the 96-well plate (50 µL/well). For a kinetic reading immediately start measuring fluorescence intensity at Ex/Em=490 nm/520 nm (FlexStation 384II, Molecular Devices(www.moldev.com). Continuously record data every five minutes for 30 to 60 minutes. For an endpoint reading incubate the reaction at room temperature for 30 to 60 min. Keep the plate from direct light. A stop solution can be optionally used (50 µL/well).
The fluorescence reading from the substrate control well represents the background fluorescence. Subtract this background reading from the readings of the other wells to get the fluorescence increase, measured in relative fluorescence units (RFU). For kinetic readings, plot data as RFU versus time for each sample. To convert RFU to concentration of the product of enzymatic reaction, use a fluorescence reference standard provided in the kit. Although cleavage efficiency of generic FRET peptide varies between MMPs, usually the assay can detect the activity of subnanogram quantities of most MMPs.
We compared the sensitivity of 5-FAM/QXL 520 MMP generic substrate and EDANS/DABCYL FRET substrate based on the same sequence using the MMP-13 enzyme. 5-FAM/QXL 520 substrate was cleaved significantly faster than the EDANS/DABCYL substrate (Figure 3). Based on these results, AnaSpec developed a panel of SensoLyte 520 MMP Assay Kits containing long-wavelength 5-FAM/QXL FRET substrates optimized for individual MMPs.