Stability, Hydration, and Thermodynamic Properties of RNase A Confined in Surface-Functionalized SBA-15 Mesoporous Molecular Sieves

Abstract

Mesoporous silicates (MPS) have several advantages for the immobilization of enzymes and large organic molecules. They possess well-defined pores and their surfaces can be functionalized by chemical methods. In this study, the model protein ribonuclease A (RNase A) was encapsulated in unmodified amino- and carboxy-functionalized rodlike SBA-15 with pore widths ranging from 4.0 to 5.8 nm. Differential scanning (DSC) and pressure perturbation (PPC) calorimetric techniques were employed to evaluate the stability, hydration, and volumetric properties of the confined protein. In addition, the influence of the solution pH, the surface functionalization, and cosolvents on the protein immobilization and the thermal stability of the immobilized protein are reported. The extent of stabilization depends strongly on the surface characteristics of the host, such as the charge density, and on geometric parameters, i.e., the pore size and pore volume. The addition of the chaotropic agent urea leads to an increased protein loading. Addition of the kosmotropic agent glycerol has the opposite effect. The stability of the protein RNase A confined in all the mesoporous silicates is drastically enhanced and is of the order of ΔT m ≈ 30 ± 10 °C regarding the increase in temperature stability. The highest immobilization capacity, fastest immobilization rate, and maximum thermal stability was achieved for the surface-functionalized SBA-15-COOH. The increased temperature stability is probably not only due to the entropy-driven excluded volume effect but also due to an increased hydration strength of the protein within the narrow silica pores, similar to the effects compatible osmolytes impose on protein hydration and stability. The absence of an expansivity increase of the confined protein after thermal denaturation indicates that inside the pores complete unfolding of the protein is not feasible anymore.

Dielectrophoretic behavior of PEGylated RNase A inside a microchannel with diamond‐shaped insulating posts

“…In microfluidics, the force exerted on a spherical particle when it is subjected to nonuniform electric fields —first introduced by Pohl in 1951— is known as the dielectrophoretic force and can be represented by the following equation : where ε m is the relative permittivity, r p is the radius of the particle, is the gradient of the squared electric field, and is the real part of the Clausius–Mossotti factor. In this work, this factor can be approximated by using the real electrical conductivities of the suspending medium and the particle since DC potentials are applied : …”

“…Ribonuclease A (RNase A), a nearly spherical 13.7‐kDa protein with a 1.5‐nm radius , has shown great potential for in vivo treatment of cancers , especially in its PEGylated form . However, grafting of PEG molecules to RNase A results in a heterogeneous mixture that contains mono‐PEGylated (mono‐PEG) RNase A, di‐PEGylated (di‐PEG) RNase A, and the unreacted protein that remains after reaction.…”

Small Molecules, Non-Covalent Interactions, and Confinement

“…Molecules 2020, 25, x FOR PEER REVIEW 5 of 31 example) and to employ special drying protocols for the preparation of "water-free" silica samples (for details see Brodrecht et al [183]). Since naturally-occurring porous hybrid materials like the skeleton of diatoms are based on modified silica materials consisting of silica and sillafins (polyamines) [184][185][186][187], the functionalization of mesoporous silica with peptides and peptoides [44,47,113,[175][176][177][178][188][189][190][191][192][193][194] creates controllable well-defined model systems for the in-vitro study of, e.g., biomineralization.…”

“…This understanding is only obtainable by a combination of various complementary spectroscopic, thermodynamic, computational, and general physico-chemical characterization techniques, including multi-nuclear variable-temperature solid-state NMR (SSNMR), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), small angle scattering (SAXS and SANS), thermogravimetric analysis (TGA) [ 35 ], and molecular dynamics (MD) simulations, as is shown by a number of recent papers (see, e.g., [ 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]). While X-ray diffraction techniques like XRD or SAXS reveal the ordered structures of these materials [ 48 , 49 , 50 ], nitrogen adsorption is employed to study the specific surface areas and pore diameters [ 51 , 52 ], DSC or TGA are used for the investigation of phase transitions inside the pores, NMR provides insights into the local ordering and dynamics on the molecular level, and computation interprets these results [ 35 , 53 ].…”

Chemically Modified Silica Materials as Model Systems for the Characterization of Water-Surface Interactions

“…Owing to their versatility, PMS materials are ideally suited for fundamental studies of the effect of confinement on the structural, dynamic and thermophysical properties of fluids in general, and water in particular or protein solutions such as RNASE A [27]. This interest is motivated by the desire to better understand the influence of surface forces and finite-size effects on the behavior of the substrate in the pores [28].…”