Relevance Of Proteases Research Articles

Voltammetric sensing of trypsin activity using gelatin as a substrate

The relevance of proteases in many areas such as the industry, microbiology, and clinical analysis has prompted the development of different methods for protease detection and proteolytic activity determination. Electrochemical sensors and assays are valuable techniques for their short response time, simple fabrication, high sensitivity and possibility of miniaturization. Here, we describe the determination of the proteolytic activity of trypsin by using a gelatin-embedded electrochemical redox mediator the (4-((4-aminophenyl)imino)-2,6-dimethoxycyclohexa-2,5-dien-1-one). The peak current (ip) was used as the analytical signal and measured by means of alternating current voltammetry (ACV). Upon exposure to different concentrations of trypsin, a decrease in the ip expressed as the relative change was observed due to the release of the redox mediator in solution. A linear response range (5–80 µg/mL) was obtained, this trend was further corroborated using standard techniques such as the azo-casein, and the gel diffusion assay. A significant and negative correlation between these assays and the trypsin activity measured as the ip relative change was observed.

Proteolysis to Identify Protease Substrates: Cleave to Decipher.

Proteolysis is an irreversible post-translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding the mechanism of proteolytic activity, the identity, and the role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, the authors recapitulate the library screening, proteomics, and bioinformatics based approaches that have been employed for the identification of protease substrates.

Relevance of protease “inhibitor” to the ATP-ubiquitin proteolytic system

ATP-dependent proteolysis in reticulocyte extracts is stimulated by ubiquitin, a polypeptide which is covalently conjugated to proteins. It has been proposed that ATP and ubiquitin act by repressing an inhibitor of an ATP-independent protease, rather than by conjugation to substrate proteins [Speiser, S. and Etlinger, J.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80 , 3577–3580] . We find that the inhibitor preparation used by these authors contains a positively required factor of the ATP-ubiquitin proteolytic system, which can be separated from two types of protease inhibitors by gel filtration chromatography. The following obervations indicate that the “inhibitors” are endogenous protease substrates which compete with the labeled substrate: (a) inhibition is competitive with exogenous substrate; (b) inhibition is abolished by a preincubation of “inhibitor” with protease prior to the addition of labeled substrate. These findings are not consistent with the notion that the inhibitors play a regulatory role in the ATP-ubiquitin proteolytic pathway.