Kinetics of Proteolytic Reactions in Nanoporous Materials

Abstract

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.

Interference-blind microfluidic sensor for ascorbic acid determination by UV/vis spectroscopy

“…The A c c e p t e d M a n u s c r i p t 9 confined space of the microfluidic channel also contributes to the high efficiency of the reaction. [25] The serpentine microfluidic channel is very long with many running-track like structures that favors the fluctuation of substrate solution in the microfluidic channel (Fig. A.…”

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

“…The amino groups on the surface of NH 2 -MOSF make the in-pore local condition appropriate to maintain tryptic activity under any bulk buffer conditions. The ultrafast reaction rate is attributed to the nano-confinement effect and the in-situ enrichment of proteins and enzymes from bulk solutions into the nanopores of NH 2 -MOSF [16,23].…”

“…With theoretical predictions and experimental validations, it is demonstrated that the porous nanomaterials efficiently accelerate biochemical reactions because they can enrich reactants into their inner pores (enrichment effect), and confine reactions in nanoscale (nanoconfinement effect) [16,17]. Macroporous materials with the pore diameters of $100 nm are currently proven as the perfect matrices to accelerate proteolysis rate, since they can provide optimum extraction and confinement to proteins (several to tens of nanometer in diameter) compared to mesoporous materials with smaller pores that give steric inhibition [15,[18][19][20].…”

Nanomaterial-Assisted Laser Desorption Ionization for Mass Spectrometry-Based Biomedical Analysis

“…Specifically, macroporous silica or titania can be used to immobilize enzymes for fast on-plate protein digestion [95][96][97], which can provide promising platforms for future tissue imaging of large biomolecules of interest.…”