A Method in Enzymology for Measuring Hydrolytic Activities in Live Cell Environments

Abstract The capability of determining the physiologic role(s) of cellular enzymes requires probes with access to all intracellular and extracellular environments. Importantly, reporter molecules must be able to cross not only the plasma membrane but also enter organelles inside live cells without disturbing the physiologic integrity of the system under study. Additionally, each enzyme must recognize a probe by the same linear and conformational characteristics as it would a physiologic substrate or inhibitor. This chapter focuses on the design and use of cell‐ and tissue‐permeable fluorogenic protease substrates. Their applications, which are far‐reaching, include measurements for apoptosis, cytotoxicity, inflammation, cancer metastasis, and viral infections such as HIV. Recently, substitution of amino acids with nucleotides in the probe backbone has allowed measurements of nuclease activities and hybridization of oligonucleotides inside live cells and an example thereof is presented.