Determination of Caspase-3 Activity and Its Inhibition Constant by Combination of Fluorescence Correlation Spectroscopy with a Microwell Chip

Caspase-3 is a key enzyme executing apoptosis during ontogenesis and homeostasis of multicellular organisms, and is a very important and potential drug target in treatment of apoptosis disturbance. So far, no commercial drugs for caspase-3 are available, and it is urgently necessitated to develop an effective method for caspase-3 activity assay and its inhibitor screening. In this paper, we propose a new method for determination of caspase-3 activity and its inhibition constant by combining single molecule fluorescence correlation spectroscopy (FCS) with a microwell chip. Its principle is based on measurement of the enzyme reaction kinetics and homogeneous detection of the reaction product by FCS. This system can reduce the requirement sample volume to 1 μL level. The caspase-3 substrates are doubly labeled with fluorophore and biotin, the enzyme reaction can be quickly terminated in the presence of streptavidin, and the reaction products can be selectively detected by FCS. We established the model of cas...