ABSTRACT
While protease inhibitor cocktails have been in use for some time,1 there are few rigorous studies examining their effect on proteolysis and very few concerned with proteolytic degradation during the processing of material for analysis or during purification.2 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 Nagase3 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, t1/2, inhibition constants, etc.), and enzyme inhibition is of particular importance. For example, with a reversible enzyme inhibitor (such as benzamidine), if the Ki value is 100 nM, a 100 µM concentration of inhibitor would be required to decrease protease activity by 99.9%. Salveson and Nagase3 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,4 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.5 The investigator is also advised to consider the modification of proteins and other biological compounds by protease inhibitors in reactions not associated with proteases such as the modification of tyrosine by DFP or PMSF.6 In addition, some of the protease inhibitors such as DFP and PMSF are subject to hydrolysis under conditions (pH ≥ 7.0) used for modification. For those unfamiliar with the history of DFP, DFP is a potent neurotoxin (inhibitor of acetyl cholinesterase) and should be treated with considerable care; a prudent investigator has a DFP repair kit in close proximity (weak base and pralidoxime-2-chloride [2-PAM]). Given these various issues, it is critical to validate that, in fact, the sample is being protected against proteolysis.
