CtsK activity was determined using either a fluorogenic substrate in a microtiter plate format or a colorimetric substrate in a cuvette method. For routine analysis of CtsK activity (such as during autoactivation process), the colorimetric substrate was used. The reaction mixture consists of 500 ng of mature cathepsin K, 40 µg z-Phe-Arg-pNA (Bachem) in 150 µl CtsK digestion buffer (100 mM sodium acetate, 5 mM DTT, 5 mM EDTA at pH 5.5). A405 measurements were taken in a spectrophotometer (BioMate 3, Thermo Fisher) for 5 min with 1 min intervals. To examine the inhibitory effects of HS, Cst3 inhibition of Ctsk, and the stability of CtsK, the fluorogenic substrate (z-Leu-Arg-AMC) was used. The reaction mixture consists of ~200 ng mature cathepsin, 5 µg z-Leu-Arg-AMC in 150 µl CtsK digestion buffer. The fluorescence signal (excitation: 370 nm, emission:450 nm) was measured on a fluorescence plate reader (Molecular Devices) for 15 min with 1 min intervals.
For digestion of the extracellular domain of human RAGE (soluble RAGE, or sRAGE), 5 μg sRAGE was digested by 20 ng mCtsK in CtsK digestion buffer at 22 °C in the presence of absence of 12mer for up to 20 mins.
For digestion of the extracellular domain of human RAGE (soluble RAGE, or sRAGE), 5 μg sRAGE was digested by 20 ng mCtsK in CtsK digestion buffer at 22 °C in the presence of absence of 12mer for up to 20 mins.