Hydration and Confinement Effects on Horse Heart Myoglobin Adsorption in Mesoporous TiO₂

Abstract

Despite the intensive research on protein adsorption in mesoporous materials, the effect of (de)­hydration and confinement on the adsorbed protein’s stability and activity is poorly understood. In this paper, we study the effect of differences in structural features (pore size) and drying time on the adsorption and structural stability of horse heart myoglobin (hhMb) on mesoporous titanium dioxide. Infrared spectroscopy (DRIFT) and thermal analysis (TGA) coupled to a quadrupole mass spectrometer (TGA–MS) were used to evaluate the impact of the confinement in different pores and hydration on the myoglobin secondary structure. Electron paramagnetic spectroscopy (EPR) was applied to identify the changes in the heme and its close surrounding. The peroxidase-like activity of myoglobin toward 2,2′-azinobis­(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide allowed us to detect changes in the protein activity after adsorption in pores with different sizes and drying for different periods of time. The results show a clear effect of the pore size and drying time on the secondary structure of hhMb, which is confirmed by differences induced in the catalytic activity of the adsorbed proteins. Therefore, we recommend evaluating the effect of both hydration and confinement in future applications involving biomolecule adsorption in porous matrices.

Understanding the Role of Surfactants in the Interaction and Hydrolysis of Myoglobin by Zr‐MOF‐808

“…From an initial concentration of 0.02 mM, only 2.04 μM HHM remained in solution after 10 minutes when no surfactant is present, indicating an adsorption of around 90 %. As has previously been found with Mb adsorption into mesoporous TiO 2 , Mb has a hydrophilic external surface, and hydrophobic regions within the globular structure, making the interaction with the MOF highly favorable [47] . The hydrophilic regions can interact with the OH groups present on the Zr 6 clusters of MOF‐808, which promotes unfolding of the protein, allowing the hydrophobic regions to further interact with the hydrophobic pores of MOF‐808 [48] …”

“…MOF‐808 in water (70 μg/mL) gave a narrow size distribution with hydrodynamic radius d H of 190–295 nm (Figure S13A). Myoglobin (0.06 mM) in water gave two peaks (Figure S13B) which correspond to the hydrodynamic diameter (d H ) of myoglobin 4.8 nm and a trace amount of protein aggregate of 68 nm [47] . The measurements preformed immediately after mixing of myoglobin and MOF‐808 in water, showed both species present individually, however, after incubation at 40 °C for 24 h, myoglobin monomers are no longer present in solution, and an increase of the hydrodynamic radius of MOF‐808 was observed (Figure 7A).…”

“…Myoglobin (0.06 mM) in water gave two peaks (Figure S13B) which correspond to the hydrodynamic diameter (d H ) of myoglobin 4.8 nm and a trace amount of protein aggregate of 68 nm. [47] The measurements preformed immediately after mixing of myoglobin and MOF-808 in water, showed both species present individually, however, after incubation at 40 °C for 24 h, myoglobin monomers are no longer present in solution, and an increase of the hydrodynamic radius of MOF-808 was observed (Figure 7A). This shows that HHM is adsorbed to the surface of the MOF, increasing the particle size and size distribution of the MOF particle.…”

“…As has previously been found with Mb adsorption into mesoporous TiO 2 , Mb has a hydrophilic external surface, and hydrophobic regions within the globular structure, making the interaction with the MOF highly favorable. [47] The hydrophilic regions can interact with the OH groups present on the Zr 6 clusters of MOF-808, which promotes unfolding of the protein, allowing the hydrophobic regions to further interact with the hydrophobic pores of MOF-808. [48] This process occurs within the first 10 minutes, and the protein remains bound to the MOF at similar levels for at least 48 h. Similar behavior is seen when MOF-808 and HHM are incubated with 0.5 % TX-100 and 0.5 % CHAPS, showing around 91 % of Mb is adsorbed after 10 minutes.…”

Hydration of TiO2 Facets Regulates As(III) Adsorption: DFT and DRIFTS Study

“…Surface hydration is the very first step and is of paramount importance in solid–water interfacial reactions. − For example, the bonded water molecules on the {001} facet break Ti–O bonds on the TiO 2 surface, forming terminal hydroxyl groups with strong hydrogen bonds . In contrast, the {101} facet is quite inert, and water molecules are observed to be molecularly adsorbed on the surface .…”