Evaluation of cardosin A as a proteolytic probe in the presence of organic solvents

This investigation showed that cardosin A not only is active in media with organic solvents, cleaving the β-chain of oxidised insulin at three susceptible peptide bonds, but also maintains its specificity in all media tested. Additionally, the presence of organic solvents in the reaction media led to modifications of enzyme selectivity, which enabled the detection of intermediate products. While solvents like ethyl acetate induced a decrease in enzymatic activity, both by reducing the amount of active enzyme and presumably due to an inhibiting effect of ethyl acetate (which might compete with the substrate for the active site of the enzyme), n-hexane caused an increase in the hydrolysis velocity of one peptide bond. In view of the activity and specificity of cardosin A (which shows high preference for hydrophobic residues), it is proposed as a reliable probe for limited proteolysis in the presence of organic solvents. This may become particularly useful for structural characterisation of membrane proteins.