In the present study, we examined the secondary and tertiary structure of myoglobin (Mb) within folded sheets mesoporous material (FSM)- and Santa Barbara amorphous (SBA)-type mesoporous silicas. The Barrett–Joyner–Halenda pore diameters of SBA-type mesoporous silicas were 39, 70, and 75 Å, and that of FSM-type mesoporous silica was 40 Å. The secondary and tertiary structures of myoglobin were observed by Fourier transform infrared (FTIR) and small-angle neutron scattering (SANS), respectively. The FTIR and SANS results indicated preservation of the secondary and tertiary structures of myoglobin inside the pores of SBA-type mesoporous silicas. Adsorption of myoglobin within FSM-type mesoporous silica, however, resulted in perturbation of the tertiary structure, accompanied by partial unfolding of the secondary structure. Lower structural stability of myoglobin within the FSM-type mesoporous silica was also confirmed. These findings suggest that the Mb structure is more influenced by the inner pore surface characteristics than by geometrical pore size.

Adsorption of protein molecules into the pores of a porous material is an important process for chromatographic separation of proteins and synthesis of nanoscale biocatalyst systems; however, there are barriers to developing a method for analyzing the process quantitatively. The purpose of this study is to examine the applicability of differential scanning calorimetry (DSC) for quantitative analysis of protein adsorption into silica mesopores. For this purpose myoglobin, a globular protein (diameter: 35.2 Å) was selected, and its adsorption onto mesoporous silica powders with uniform pore diameters (pore diameters: 39 and 64 Å) was measured by adsorption assay and DSC experiments. Our results confirmed that the adsorption of myoglobin into the silica mesopores induced significant changes in the positions and areas of freezing/melting peaks of the pore water. The decrease in heat of fusion of the pore water after myoglobin adsorption could be utilized to quantify the amount of myoglobin inside the silica mesopores. The advantages of DSC include its applicability to small wet mesoporous silica samples.

We studied the stabilities of short (4- and 3-bp) DNA duplexes within silica mesopores modified with a positively charged trimethyl aminopropyl (TMAP) monolayer (BJH pore diameter 1.6-7.4 nm). The DNA fragments with fluorescent dye were introduced into the pores, and their fluorescence resonance energy transfer (FRET) response was measured to estimate the structuring energies of the short DNA duplexes under cryogenic conditions (temperature 233-323 K). The results confirmed the enthalpic stability gain of the duplex within size-matched pores (1.6 and 2.3 nm). The hybridization equilibrium constants found for the size-matched pores were 2 orders of magnitude larger than those for large pores (≥3.5 nm), and this size-matching effect for the enhanced duplex stability was explained by a tight electrostatic interaction between the duplex and the surface TMAP groups. These results indicate the requirement of the precise regulation of mesopore size to ensure the stabilization of hydrogen-bonded supramolecular assemblies.

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