FIGURE 5.

Effect of chondroitin 4-sulfate on the conformation of cathepsin K. A, stoichiometric-dependent changes in pro-hrCatK (1 μm) in the presence of C4-S was monitored by the intrinsic fluorescence of tryptophan moieties. Measurements were taken in acetate buffer at pH 5.0 and with salt (100 mm NaCl, 1 mm CaCl₂) and at 22 °C where autoactivation is insignificant over the duration of titration and measurements. The reaction was mixed after each addition of C4-S and allowed to equilibrate for 2 min. B, stoichiometry-dependent autoactivation of 4 μm pro-hrCatK in the presence of C4-S. The reaction mixture was incubated at 37 °C for 6 h also in acetate buffer at pH 5.0 and salt. C, fluorescence changes of the CatK inhibitor L-235 (6 μm) in the presence of C4-S (6 μm) and pro-hrCatK (6 μm) or preactivated CatK (hrCatK-Act). Measurements were taken at 37 °C with excitation at 315 nm using a Micromax 384 plate reader.