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PDQ™ Protease Assay Dave Samaroo, Rosalind Ramsey, and Sheldon E. Broedel, Jr. Athena Environmental Sciences, Inc. 1450 South Rolling Road, Baltimore, MD 21227 The colorimetric PDQ™ Protease Assay was first introduced in 1996. It has since become a routinely used assay for measuring protease activity in process samples or for certifying reagents as protease-free. However, while the sol-gel substrate matrix allows for a one-step assay with nanogram detection limits, the substrate matrix only has a three month shelf-life. Alternative matrix formats, including the use of fluorescent substrates, have not yielded a more robust assay. Here we describe development of a protease assay using a fluorescent protease substrate in combination with metal nanoparticle coated microtiter wells. The metal-enhanced fluorescent signal permitted the development of an assay with a detection limit more than 100fold lower than the original assay with detection limits below 10 pg for certain proteases. An accelerate stability study suggested that the shelf-life of the substrate is at least one year at 4ºC. Further, the assay is adaptable to high-throughput formats. Introduction Importance: Proteases are well known commercial enzymes that are exploited in the detergent, food, pharmaceutical, diagnostics, and fine chemical industries. Their use as detergent additives is the largest application of industrial enzymes in terms of volume and value. Interest in proteases has increased with the realization that they play a critical role in a variety of diseases. Modifications in proteolytic systems may trigger multiple pathological conditions such as cancer, neurodegenerative disorders, and cardiovascular diseases. In industrial settings, the formation of biofilms is an economic problem and proteases are often used to remove and clean these biofilms from surfaces. Further, researchers and manufacturers of high valued proteins are concerned with undesirable degradation by contaminating proteases. All of these applications require the use of a quantitative measure of protease activity. Background of PDQ™ Protease Assay: The PDQ™ Protease Assay is a colorimetric assay that was designed to simplify the measurement of protease activity. The assay format eliminates the separation steps often used in historic protease assays thereby allowing for a single step assay. The assay is a vial-based system consisting of cross-linked sol-gel composed of albumin, azoalbumin, and gelatin. This substrate matrix is susceptible to a wide range of enzymes commonly used in industry and research. However, the manufacture of the assay is labor intensive and the product suffers from a short shelf-life. Further, the detection limit is between 100 ng and 10 µg depending on the enzyme. Objective of new format: To develop an assay that has a lower detection limit, is adaptable to high throughput systems and is more cost effective to manufacture, we evaluated the use of fluorescence tagged protease substrate. The new format consists of a 96well plate using metal-enhanced nano-particles to increase the fluorescent signal. The fluorescently labeled substrate is immobilized on the surface of the metal nano-particles and protease activity is quantified by measuring the amount of residual fluorescence remaining in the well or the amount of fluorescence released into the buffer. This allows for a one step procedure with reaction conditions that are matrix-independent including the use of opaque samples. Figure 1. Metal nano-particle coated plates increase the fluorescence signal yielding a more sensitive assay. Method: The wells of a Maxisorb™ (Nunc) and QuantaWell™ (Athena) plate were coated with 100 µg of fluorescently labeled BSA. Duplicate wells were incubated with 0.1 ml of trypsin at different concentrations ranging from 50 to 0.05 BAEE Units/ml and the residual level of fluorescence measured after 1 hour incubation at 37ºC. Residual LOD ~ 0.01 Unit/ml LOD ~ 1 Unit/ml Method: QuantaWells™ (Athena) were coated as in Fig. 3. Duplicate wells were incubated with 0.1ml of trypsin at concentrations ranging from 102 to 10-3 BAEE Units/ml and the amount of fluorescence released (left panel) and residual (right panel) measured after 1 hour incubation at 37ºC. Figure 5. The substrate matrix coated on QuantaWells™ was stable at elevated temperature for 48 days. This indicates a product shelf-life of 1 year at 4ºC. Figure 2. The signal is proportional to the amount of substrate coated on the well. Released Substrate using 40 BAEE/ml Trypsin 45000 40000 35000 30000 25000 20000 Trypsin Conc. (Units/ml) 15000 Methods: QuantaWell™ strip wells were coated as in Fig. 3 and stored at 50ºC. At the times indicated, a duplicate set of strips were reacted with 0.1 ml trypsin and the amount of residual fluorescence measured as in Fig. 3. 10000 5000 0 0.0 200.0 400.0 600.0 800.0 1000.0 Coating Concentration (µg/ml) Method: A QuantaWell™ plate was coated with different concentrations of fluorescently labeled BSA. Duplicate wells were incubated with 0.1 ml of 40 BAEE units/ml trypsin for 1 hour at 37ºC and the amount of residual (not shown) and released fluorescence measured. The selected coating concentration for further development was 500 µg/ml (50 µg per well). Figure 6. The activity of a range of different type of proteases can be measured using the fluorescent PDQ™ Protease Assay. Figure 3. Enhanced fluorescence assay exhibited a 100-fold lower detection limit compared to the standard colorimetric assay. Methods: QuantaWells™ were coated as in Fig. 3 and duplicate wells incubated at 37ºC with 0.1 ml of seven different proteases at concentrations ranging from 100 µg/ml to 10 pg/ml. After 1 hour, the amount of residual fluorescence was determined. References: Kirk, O., Borchert, T., & Fuglsang, C. (2002). Industrial enzyme applications. Current Opinion In Biotechnology, 13(4), 345-351. doi:10.1016/s0958-1669(02)00328-2. Lopez-Otin, C., & Bond, J. (2008). Proteases: Multifunctional Enzymes in Life and Disease.Journal Of Biological Chemistry, 283(45), 30433-30437. doi:10.1074/jbc.r800035200. Selan, L., Berlutti, F., Passariello, C., Comodi-Ballanti, M., & Thaller, M. (1993). Proteolytic enzymes: a new treatment strategy for prosthetic infections?. Antimicrobial Agents And Chemotherapy, 37(12), 2618-2621. doi:10.1128/aac.37.12.2618 Royer, G. P. and Broedel, Jr., S. E. (1996). One Step Protease Assay Technical Brief. Retrieved 8 January 2015, from http://www.athenaes.com/tech_brief_protease.php (accessed 8 Jan 2015). Aslan, K., Gryczynski, I., Malicka, J., Matveeva, E., Lakowicz, J., & Geddes, C. (2005). Metalenhanced fluorescence: an emerging tool in biotechnology. Current Opinion In Biotechnology, 16(1), 55-62. doi:10.1016/j.copbio.2005.01.001. Figure 4. Measuring the residual fluorescence permitted detection of lower amounts of protease activity. Released RFU Abstract Summary and Conclusions: Methods: A QuantaWell™ plate was coated with 50 µg fluorescently labeled BSA. Duplicate wells were incubated with 0.1 ml of 100 to 0.001 BAEE units/ml trypsin and the residual fluorescence measured after 1 hour at 37ºC. The resulting calibrator curve (right panel) was compared to the calibrator curve generated by the colorimetric assay (left panel). The Fluorescent PDQ™ Protease Assay using metal nano-particle coated QuantaWell™ microtiter plates exhibits: • Lower detection limits by two or more orders of magnitude compared to the original assay. • Ability to measure a broad spectrum of proteases. • Lower manufacturing costs. • Increased product shelf life.