title: Reverse zymography alone does not confirm presence of a protease inhibitor.
abstract: Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.
authors: Division of Structural Biology and Bioinformatics, CSIR-IndianInstitute of Chemical Biology, 4, Raja S.C. Mallick Road, Jadavpur, Kolkata 700032, India.
authors: Sangita
authors: S
authors: Dutta
authors: Debasish
authors: D
authors: Bhattacharyya
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D004792:Enzyme Precursors / Q000737:chemistry*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry*; D059748:Proteolysis
chemical_list: D004792:Enzyme Precursors; D011480:Protease Inhibitors; D010447:Peptide Hydrolases