Avoiding proteolysis during the extraction and purification of active plant enzymes

The aim of this article is to discuss approaches to diagnose and prevent unwanted proteolysis during extraction and isolation of active enzymes from plant tissues. Enzymes are protein catalysts that require great care during sample processing in order to ensure that they remain intact and fully active. Preventing artifactual enzyme modifications ex planta is of utmost importance in order to obtain biologically relevant data. This is particularly problematic following enzyme extraction from plant tissues, which relative to microbes or animals contain relatively low protein amounts coupled with high concentrations of vacuolar proteases. Although cytoplasmic enzymes are not directly accessible to vacuolar proteases owing their physical segregation into different subcellular compartments, this compartmentation is destroyed during cell lysis. Unwanted proteolysis by endogenous proteases is an insidious problem because in many cases the enzyme of interest is only partially degraded and retains catalytic activity. This can not only lead to erroneous conclusions about an enzyme’s size, subunit structure and post-translational modifications, but can also result in striking changes to its kinetic and regulatory (i.e. allosteric) properties. Furthermore, the routine addition of class-specific protease inhibitors and/or commercially available (and expensive) protease inhibitor cocktails to extraction and purification buffers does not necessarily preclude partial proteolysis of plant enzymes by endogenous proteases. When antibodies are available, plant scientists are advised to employ immunoblotting to diagnose potential in vitro proteolytic truncation of the enzymes that they wish to characterize, as well as to test the effectiveness of specific protease inhibitors in overcoming this recurrent issue.