Pressure Cycling Technology (PCT) has been proven to accelerate enzymatic protein digestion. The positive effect of PCT on trypsin digestion is well established for improved sequence coverage, higher recovery and significantly reduced digestion times [1-5]. Additionally, the enhancing effects of PCT on the activity of other enzymes, including Proteinase K, PNGase F, Glu-C, Lys-C, and lysozyme have been reported [6-12].
Pressure induces protein denaturation, but the pressure-perturbed proteins assume conformational forms that are different from those caused by thermal or chemical treatment . Pressure-induced denaturation of the substrate proteins leads to better enzyme access to previously inaccessible target sites. This, in turn, results in improved and accelerated digestion, as long as the level of pressure that is applied is below the level at which the enzyme itself is denatured and inactivated. In addition, under certain conditions, hydrostatic pressure can have a positive effect on enzyme activity, apparently independent of substrate conformation.
Here we report that PCT can increase specific chymotrypsin activity and accelerate the digestion of model proteins in solution. The goal of this work is to examine the effect of PCT on chymotrypsin activity and to provide the user with the best set of starting conditions for pressure-enhanced chymotrypsin digestion.