The positive effect of Pressure Cycling Technology (PCT) on trypsin digestion is well established [1-4], and has been shown to result in improved sequence coverage, higher recovery and significantly reduced digestion times. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Lys-C, Proteinase K, PNGase F, chymotrypsin, Glu-C, thermolysin, and lysozyme has been reported [5-12].
Pressure, heat, and many different chemicals can be used to denature proteins, but the pressure-perturbed proteins assume conformational forms that are different from those caused by thermal or chemical treatments . Pressure-induced denaturation of substrate proteins leads to better access of enzymes to previously inaccessible, or poorly accessible, 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, independent of substrate conformation.
Here we report that protein digestion by elastase enzyme is accelerated when the enzyme reaction is carried out under pressure cycling conditions. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced Elastase digestion.