In vitro, MENT is a potent inhibitor of the papain-like cysteine proteases cathepsins K, L, and V. Stoichiometry of inhibition (SI) and second-order association rate constants (ka) can be assessed using the fluorogenic substrate N-Cbz-Phe-Arg-methylcoumarin as described elsewhere (Irving et al., 2002; McGowan et al. (2006; Ong et al., 2007) and are further outlined in Chapter 11. Enzyme assays between MENT and target cathepsins were performed at 30 °C. °C Assay buffer (0.1 M acetate, pH 5.5 , 1 mM EDTA, 0.1%, w/v Brij-35, 10 mM cysteine), all cathepsins were passed in cathepsin buffer (0.1 M acetate, 1 mM EDTA, at room temperature) before use At least 20 min (0.1%, w/v Brij-35, 0.02%, w/v sodium azide, 10 mM cysteine, pH 5.5). Active enzyme concentration was determined by trans-epoxysuccinyl-1-leu Aminoamide titration determination - (4-guanidino)-butane (E-64).
3.2 Natural acid PAGE
The oligomeric state of MENT (and the complex between MENT and the target protease) can be visualized using a 12% native acid gel protocol (Zhou et al., 2001) with methyl green used as a tracking dye and the protein towards the negative pole. The electrode buffer contained 40 mM β-alanine, adjusted to pH 4.0 with acetic acid. Electrophoresis is usually performed at room temperature. Separation of MENT proteins and protein complexes was successful when the gel was run at 80 V for approximately 4-5 hours. Natural acidic PAGE gels were prepared fresh on the day of use. Proteins were visualized by staining with Coomassie Brilliant Blue R-250.

Intrinsic tryptophan fluorescence assesses conformational changes
Potential cofactor-induced conformational changes in MENT proteins can be studied by monitoring changes in intrinsic tryptophan fluorescence in the presence and absence of the cofactor. Incubate MENT protein (0.2 μM) and increasing cofactors in a final volume of 2 mL in 10 mM HEPES (pH 7.0) containing 0.5 mM EDTA, 40 mM NaCl, and 0.1% (w/v) Brij-35. Experiments were repeated three times. Scan emission ≥5 times at 60 nm/min in the range 300–400 nm using excitation and emission slit widths between 5 and 8 nm. The emission spectra of buffers in the presence and absence of cofactors used as controls were subtracted from the protein emission spectra. Always use acrylic cuvettes to avoid adsorption to the cuvette walls trestolone acetate
When using DNA-containing cofactors, use an excitation wavelength of 295 nm to minimize internal filter effects for the measured fluorescence, and monitor the DNA absorbance at the excitation wavelength and not exceed 0.1 for any sample. Fluorescence quenching experiments were performed using increasing concentrations of acrylamide (0–0.5 M) in the presence and absence of the DNA cofactor. From the recorded titration spectra, the degree of quenching and the accessibility of tryptophan residues can be calculated based on the Stern-Volmer plot previously described by Lehrer