Investigation of Pressure-Enhanced Trypsin Proteolysis
Pressure Cycling Technology and Pressure Effects on Biological Macromolecules
Pressure is a well-understood thermodynamic parameter that affects enzyme activity and protein conformation. High pressure can weaken hydrophobic interactions but can enhance electrostatic ones [1, 2]. The main pressure effects on biological macromolecules are attributed to perturbation of the interactions with solvent, leading to reversible partial protein denaturation, weakening of lipid bilayers and dissociation of multimeric protein complexes . Pressure-perturbed proteins have been shown to assume conformational forms drastically different from those resulting from thermal or chemical treatment alone .
Pressure Cycling Technology (PCT) uses alternating high hydrostatic pressure to facilitate thermodynamic perturbation of molecular interactions. PCT has been shown to modulate enzymatic proteolysis and deglycosylation, and improve sequence coverage. In addition, this system offers specific advantages for tissue and cell lysis, and improved recovery of hydrophobic molecules such as integral membrane proteins [5, 6].
While most of the published work to date is based on empirical optimization of high pressure extraction and digestion methods [6-9], we present part of a systematic study investigating the impact of hydrostatic pressure on trypsin specificity. Our goal is to understand the relationship between cleavage site environment and enzyme activity, as a function of high pressure. Our intent is to systematically investigate high pressure effects on proteases and substrate proteins within the context