Cleavage of pro-tumor necrosis factor alpha by ADAM metallopeptidase domain 17: a fluorescence-based protease assay...

A fluorescence-based Adam 17 activity assay that cleaves pro-tumor necrosis factor alpha (pro-TNFα) protein substrate has been developed. The key to the assay was site-specific labeling of a fluorescence dye to the N-terminal end of the substrate protein, which was achieved by the protein ligation method. The protease cleavage reaction was monitored by fluorescence polarization. This homogeneous assay allows reaction progress to be recorded kinetically in real time. The results were validated by gel electrophoresis and high-performance liquid chromatography. As expected, the reaction could be inhibited by an ADAM metallopeptidase domain 17 (Adam 17) active site inhibitor. Interestingly, the reaction rate of pro-TNFα cleavage by Adam 17 was also reduced by a small molecule binding to pro-TNFα protein, the substrate of the reaction. This small molecule, however, did not affect the activity of Adam 17 to its peptide substrate. These results demonstrate that this natural protein substrate-based fluorescent assay was able to identify the inhibitor binding to substrate protein in addition to that binding to the protease itself. Comparing this protein substrate with a short peptide substrate, the activity of Adam 17 showed different pH profiles. With pro-TNFα the optimal pH was approximately 7.4, whereas with the peptide substrate the optimal pH was higher than 9.0.