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

Abstract

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.