Mechanisms

Proteases (also termed “proteolytic enzymes” “peptidases” or “proteinase”) attack their substrates using 2 approaches. Exopeptidases remove single residues or dipeptides sequentially from either the N-terminus or C-terminus depending on the specificity of the particular protease, whereas endopeptidases make one or mroe cleavages within the protein to break it down into smaller polypeptide fragments. A natural consequence of protein cleavage is the generation of a new N-terminus if the cleavage is carried out by an aminopeptidase, a new C-terminus if the hydrolysing enzyme is a carboxypeptidase, or a new N-termini and C-termini if an endopeptidase is involved. These termini are antigenically distinct form the intact protein. Because these new termini are antigenically distinct from the intact protein, it is possible to generate antibodies that react with such new epitopes can be generated. The general term “anti neoepitope antibody” has been used to describe such antibodies.

Applications and Functions

Proteolytic enzymes regulate protein processing and intravellular protein levels by removing abnormal and damaged proteins from the cell as well as play a defensive role against herbivores, inects and pests. In addition to their biological roles, proteases have also been exploited commercially in food, leath, texitle, and detergent industrities because of their broad substrate specificites and activities over a wide range of pH, temperature and other denaturing conditions. In the detergent industry, for example, proteases are used to improve the cleaning efficiency of laundry detergents. (Kumari, J. Agric. Food Chem. 2010, 58, 8027-8034).

Although necessary for proper function during inflammation and remodeling, proteases can cause serious tissue damage if improperly regulated. For example, extracellular matrix degradation is an underlying feature of many degenerative diseases, such as arthritis. Although the hydrolysis of peptide bonds is an integral part of most physiological and pathological processes, knowledge is often lacking as to which peptide bonds are cleaved, in which protein substrates, in which order, and by which proteolytic enzymes (Mort, J Clin Pathol, 1999, 52 11-18).

Common Proteases

Clostripain (AKA “endoproteinase Arg-C”): is a two chain proteinase that can be isolated from Clostridium histolyticum. It has been shown to have both proteolytic and amidase/esterase activity and has an optimal pH range of 7.6-7.9. Clostripain preferentially cleaves at the carboxyl group fo arginine residues, however the lceavage of lysyl bonds has also been reported.. While Clostripain has been shown to accept substrates contianing Lys instead of Arg, reaction rates are low in comparison to reactions with Arg contining substrates. Clostripain clevage is often used in biomedical and biotechnological applications. For example, clostripain cleavage include peptide mapping, sequence analysis, cell isolation, hydrolysis/condensation of amide bonds and peptide synthesis. Clostripain may be used in order to cleave of tags. 

Papain: is a plant dervied sulfhydryl protease which is commonly used to digest IgG antibodies into either Fab or F(ab’2.)2 fragments, depending on whether L-cysteine is present or absent during the reaction, respectively.