ABSTRACT
It is usually assumed that proteolysis can be a problem and protease inhibitors or protease inhibitor cocktails are usually included as part of a protocol without the provision of justification. There are several excellent review articles in this area. Salveson and Nagase
discuss the inhibition of proteolytic enzymes in great detail including much practical information that should be considered in experimental design. The discussion of the relationship between inhibitor concentration, inhibitor/enzyme binding constants (association constants, binding constants, t
, inhibition constants, etc.), and enzyme inhibition is of particular importance. For example, with a reversible enzyme inhibitor (such as benzamidine), if the K
value is 100 nM, a 100 µM concentration of inhibitor would be required to decrease protease activity by 99.9%. Salveson and Nagase
also note the well-known differences in the reaction rates of inhibitors such as DFP and PMSF with the active site of serine proteases. DFP is much faster than PMSF with trypsin but equivalent rates are seen with chymotrypsin. PMSF is included in commercial protease inhibitor cocktails because of its lack of toxicity compared to DFP; 3,4-dichloroisocoumarin (3,4-DCI), as described by Powers and colleagues
, is faster than either DFP or PMSF. Also enzyme inhibition occurs in the presence of substrate (proteins), which will influence the effectiveness of both irreversible and reversible enzyme inhibitors. In addition, some protease inhibitor cocktails include both PMSF and benzamidine. Benzamidine is a competitive inhibitor of trypticlike serine proteases and slows the rate of inactivation of such enzymes by reagents such as PMSF.
