Kinetics of proteolytic reactions in nanoporous materials.

Proteolysis with proteases preloaded within nanopores of porous material is a very fast process, where proteins can be digested in minutes compared to the conventional bulk enzyme reactions taking place over hours. To model this surprising phenomenon, a modified sequential proteolytic mechanism has been developed to simulate the kinetics of the reaction. Digestion of myoglobin was used as an example to show the high efficiency of the in-nanopore enzymatic reaction, while angiotensin 1 and ACTH (1-14) were selected as model peptides to validate the theoretical considerations. The proteolytic peptides were quantified by capillary electrophoresis and sequenced by mass spectrometry using bottom-up strategy. The simulation clearly shows that the major factor for the very fast digestion kinetics observed stems from a peptide confinement effect, where the generated peptides are trapped within a confined space for further proteolysis to the final products. On the other hand, the ingress and diffusion of the proteins into the porous cavity can accelerate or limit the first proteolytic step requiring the encounter between the substrates and enzymes. The present model can be widely applied to different enzyme catalyzed reactions for high-throughput protein profiling, and can promote the study of enzyme reactions occurring inside the cell.