Anal. Methods, 2016, 8, 6270-6276. DOI: 10.1039/C6AY01256F

A colorimetric protease activity assay method using engineered procaspase-3 enzymes

Dokyung Yanga, Hyeon Ji Parka and Tae Hyeon Yooa,b

aDepartment of Molecular Science and Technology and bDepartment of Applied Chemistry and Biological Engineering, Ajou University, Suwon, Korea



Proteases have been important diagnostic indicators because of their relevance to disease states. Therefore, different types of assay methods have been developed for the accurate measurement of protease activity. In this study, we report a colorimetric protease activity assay method for matrix metalloproteinase 2 (MMP2) using an engineered procaspase-3. The tailor-made procaspase-3 is activated by MMP2, and the active enzyme produces a yellow absorbance signal by hydrolyzing a colorimetric caspase-3 substrate. Optimization of this assay system permitted the assessment of MMP2 activity at a picomolar concentration range even though the assay system is based on absorbance. The enzymatic reaction cascade leads to signal amplification and contributes to the high sensitivity of the assay method. The sensitive and simple assay method developed in this study can be developed as a diagnostic tool for MMP2-associated diseases. Additionally, the design strategy of procaspase-3 can be generalized, and the technology platform is expected to be easily adapted for other proteases.



Proteases play important roles in the regulation and control of various biological phenomena, and thus abnormal activities of these enzymes have been implicated in diverse diseases including cancer, cardiovascular disease, Alzheimer’s disease, and inflammatory diseases.1-3 In addition, the pathogenic roles of proteases in viral infection pathways have been well documented.4, 5 Involvement of proteases in these various disease states has led to the development of assay techniques that use proteases as diagnostic indicators. Among the various approaches to analyze proteases associated with disease states, engineering protein switches, which can be activated by the proteases of interest, have recently been attempted for several enzymes yielding detectable signals because of a potential to develop highly sensitive assay methods via signal amplification.6-8 In this published work, we engineered a procaspase-3 enzyme that can be activated by MMP2 and developed a sensitive colorimetric MMP2 activity method using the tailor-made procaspase-3.

Caspase-3 is a member of the cysteine–aspartic acid protease family and plays a central role in the cellular apoptotic pathway.9, 10 The enzyme exists in the cytoplasm as a proenzyme that can be activated via cleavage at the two Asp residues (D28 and D175) by other caspases such as caspase-8 and caspase-9. This proteolytic processing produces two subunits, large (17 kDa) and small (12 kDa), that dimerize to form the active enzyme; two identical heterodimers associate to form a tetramer of mature caspase-3. The MMP2 cleavable sequence (GPL¯GVR; the down arrow indicates the peptide bond cleaved by MMP2) extended with Gly and Ser was inserted instead of D175 into the second proteolytic processing sequence (P4-I172ETDS176-P1’) between the large and the small subunit. Since it is known not to affect the conformation of the procaspase-3 dimer and not essential for the expression, the prodomain was removed in designing these constructs.11 The construct shown in Fig. 1b exhibited the most efficient cleavage efficiency by MMP2 and the highest activity ratio of the processed enzyme to the unprocessed one.



Fig. 1 (a) Schematic illustrating the protease activity assay method developed in this study. (b) Structure of the designed procaspase-3 enzyme that can be activated by MMP2. A MMP2 cleavable linker (GGSGPL¯GVRGG) was inserted between T174 and S176 instead of D175. (c) MMP2 activity assay procedure. (d) A time-resolved substrate (Ac-DEVD-pNA) hydrolysis for samples with or without MMP2.


We developed a colorimetric assay method for MMP2 activity of samples using the designed procaspase-3 construct (Fig. 1c). Samples for MMP2 activity assay were mixed with the procaspase-3 solution, and then the caspase-3 substrate (Ac-DEVD-pNA) solution was added to the sample solution. The hydrolysis of Ac-DEVD-pNA was determined by measuring the change of absorbance at 405 nm (Fig. 1d). The limit of detection for MMP2 was as low as 52 pM with a 4 h incubation of samples with procaspase-3 and could be decreased to 6.5 pM by extending the incubation time to 16 h. In addition, the assay method was tested in biological samples including urine, E. coli lysate, and HEK 293 lysate.



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This research was supported by the National Research Foundation of Korea (2015R1A1A1A05000898, 2015R1A2A2A09001059, 2015M3D3A1A01064878, and 2014M3C1A3051460), and was also partially supported by the Ajou University research fund.



Tae Hyeon Yoo

Associate Professor

Department of Molecular Science and Technology

Ajou University

Worldcupro 206, Suwon, 16499, Korea (ROK)

Phone: +82-31-219-3543




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