Enhancing Stability and Oxidation Activity of Cytochrome c by Immobilization in the Nanochannels of Mesoporous Aluminosilicates

Abstract

Hydrothermally stable and structrurally ordered mesoporous and microporous aluminosilicates with different pore sizes have been synthesized to immobilize cytochrome c (cyt c): MAS-9 (pore size 90 A), MCM-48-S (27 A), MCM-41-S (25 A), and Y zeolites (7.4 A). The amount of cyt c adsorption could be increased by the introduction of aluminum into the framework of pure silica materials. Among these mesoprous silicas (MPS), MAS-9 showed the highest loading capacity due to its large pore size. However, cyt c immobilized in MAS-9 could undergo facile unfolding during hydrothermal treatments. MCM-41-S and MCM-48-S have the pore sizes that match well the size of cyt c (25 x 25 x 37 A). Hence the adsorbed cyt c in these two medium pore size MPS have the highest hydrothermal stability and overall catalytic activity. On the other hand, the pore size of NaY zeolite is so small that cyt c is mostly adsorbed only on the outer surface and loses its enzymatic activity rapidly. The improved stability and high catalytic activity of cyt c immobilized in MPS are attributed to the electrostatic attraction between the pore surface and cyt c and the confinement provided by nanochannels. We further observed that cyt c immobilized in MPS exists in both high and low spin states, as inferred from the ESR and UV-vis studies. This is different from the native cyt c, which shows primarily the low spin state. The high spin state arises from the replacement of Met-80 ligands of heme Fe (III) by water or silanol group on silica surface, which could open up the heme groove for easy access of oxidants and substrates to iron center and facilitate the catalytic activity. In the catalytic study, MAS-9-cyt c showed the highest specific activity toward the oxidation of polycyclic aromatic hydrocarbons (PAHs), which arises from the fast mass transfer rate of reaction substrate due to its large pore size. For pinacyanol (a hydrophilic substrate), MCM-41-S-cyt c and MCM-48-S-cyt c showed higher specific activity than NaY-cyt c and MAS-9-cyt c. The result indicated that cyt c embedded in the channels of MCM-41-S and MCM-48-S was protected against unfolding and loss of activity. By increasing the concentration of the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in ESR experiments, we showed that cyt c catalyzes a homolytic cleavage of the O-O bond of hydroperoxide and generates a protein cation radical (g = 2.00). Possible mechanisms for MPS-cyt c catalytic oxidation of hydroperoxides and PAHs are proposed based on the spectroscopic characterizations of the systems.

Proteins in Mesoporous Silicates

“…Others have also advocated that a snug fit inside the pores was desirable. [63][64][65][66] Smaller pore entrances can also improve overall catalytic activity by reducing leaching; for example, MCF materials with smaller pore entrances exhibited better stability, activity, and recycling properties than materials with larger entrances for an immobilized antibody in organic solvents. [67] However, there must be room for the substrate to have access to the protein, as limitations on the substrate diffusion rate can reduce the overall activity of the immobilized enzyme.…”

“…[67] However, there must be room for the substrate to have access to the protein, as limitations on the substrate diffusion rate can reduce the overall activity of the immobilized enzyme. [64,68] The hydrophobicity or hydrophilicity and size of the substrate will have a very strong influence on the catalytic activity of the immobilized enzyme. [46,64,66] If the substrate is hydrophobic, diffusion through the channels of a hydrophilic mesoporous silica structure may be slow.…”

“…[64,68] The hydrophobicity or hydrophilicity and size of the substrate will have a very strong influence on the catalytic activity of the immobilized enzyme. [46,64,66] If the substrate is hydrophobic, diffusion through the channels of a hydrophilic mesoporous silica structure may be slow. Larger pore sizes may compensate for this mismatch, with higher diffusion rates than for smaller pores.…”

Biodegradable mesoporous calcium–magnesium silicate-polybutylene succinate scaffolds for osseous tissue engineering

“…It has been shown that materials with a mesoporous structure promote protein unfolding and adsorbed protein density. 32 Thus, the om-CMS/PBSu substrate could promote increased FN binding by providing a larger surface area and pore volume thereby enhancing cell adhesion significantly. In addition, the hydrophilic surface of the om-CMS/PBSu is beneficial for cell attachment, spreading, and cytoskeletal organization.…”

Synthesis of self-assembled nanoporous silica for therapeutic delivery

“…However, particles with pore size significantly larger than the cargo will not benefit for the performance. Mou group 50 reported that MSNs with pore size of ~2.5 nm, which is very close to cytochrome c size, possess better capability of improving the protein hydrothermal stability and catalytic activities than porous supports with small pores (0.7 nm) or very large pores (9 nm). Recently our group 51 reported RNase A delivery using silica vesicles with different entrance size.…”