Differential Scanning Calorimetry Study on the Adsorption of Myoglobin at Mesoporous Silicas: Effects of Solution pH and Pore Size

Abstract

In the present study, pore adsorption behavior of globular myoglobin (Mb) at mesoporous silicas was examined utilizing the low-temperature differential scanning calorimetry (DSC) method. The DSC method relies on a decrease in heat of fusion for the pore water upon adsorption of Mb. The amount and structure of Mb adsorbed into the mesoporous silica were examined by DSC and optical absorption spectroscopy. The results indicated that the pore adsorption behavior of Mb strongly depended on the solution pH and pore size of mesoporous silica. For the adsorption of Mb (diameter = 3.5 nm) into mesoporous silica with narrow pores (pore diameter = 3.3 nm) at a pH ranging from 7.0 to 3.7, the penetration of both folded and denatured Mb molecules was confirmed. The folded Mb could penetrate into large mesoporous silica pores (pore diameter = 5.3 and 7.9 nm), whereas the penetration of the denatured Mb molecules was completely inhibited. The distribution of folded Mb at mesoporous silica depended on the pore size; almost all folded Mb molecules located inside mesoporous silica pores of diameters 3.3 and 5.3 nm, whereas the Mb molecules distributed at bot internal and external pore surfaces of mesoporous silica with 7.9 nm in pore diameter. These pore adsorption behaviors suggest that aggregation or stacking of the Mb molecules at the pore entrance regions of the large pores affected the pore adsorption behavior.

Structural Characterization of Proteins Adsorbed at Nanoporous Materials

“…The DSC method relies on observing the decrease in the mesopore volume upon the adsorption of protein molecules at the internal pore surface of mesoporous particles. 48,71,72 When suspension of particulate mesoporous material is applied for DSC measurement, the melting peak of the water inside the pores (pore water) appears to be far below the melting temperature of bulk water (273 K). The melting peak area (heat of fusion) of the pore water is proportional to the mesopore volume, and the change in the mesopore volume upon the adsorption of protein molecules is used to estimate the amount of protein molecules located inside the pores.…”

“…3(B)). 48,71,72 The former parameters have been extensively studied for mesoporous silica materials, 45,[50][51][52] but those for other mesoporous materials are still unclear. The DSC method hence requires some assumption on those parameters to analyze the DSC data.…”

“…However, its availability was demonstrated for the adsorption of myoglobin at mesoporous silicas with different pore sizes. 71,72 Figure 4 shows the total amount of myoglobin adsorbed at mesoporous silica particles (Atot) and the amount of myoglobin Fig. 4 Comparison of the total amount of adsorbed myoglobin (Atot) and the amount of myoglobin inside pores (Apore) for mesoporous silicas with (A) 6.9 nm and (B) 3.9 nm in pore diameters.…”

“…On the other hand, recent studies have indicated that such pore adsorption does not always take place. 71,72,78 Myoglobin (3.5 nm) molecules are effectively adsorbed inside a size-matched pore of mesoporous silica (BJH pore diameter, 3.9 nm), whereas they are preferentially adsorbed at the external surface of mesoporous silica with large pores (BJH pore diameter, 6.4 nm). 71 Even for protein molecules that are located inside the pore region, their distribution inside mesoporous material would not be unity.…”

“…74 These inhomogeneous protein distributions may be related to stacking and/or aggregation of protein molecules. 72 For further studying the structural characterization of a protein adsorbed at a mesoporous material, determining the protein distribution would be necessary to clarify any confinement effect for the structure and the function of a protein.…”