Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

Abstract

Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys 23 -Cys 63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.