Enzyme–inorganic nanoporous materials: Differential scanning calorimetric studies and protein stability

Direct assessment of the thermodynamic stabilities of enzymes bound to solids is essential to understand the factors that control bound enzyme stability. Here, the first reports of the thermodynamic stabilities of enzymes/proteins which are bound to an inorganic solid [α-Zr(HPO4)2 · H2O, abbreviated as α-ZrP] are described. The thermal denaturation of hen egg white lysozyme (Lys), met-myoglobin (Mb) and met-hemoglobin (Hb) bound to α-ZrP occurs over a wide range of temperatures (50–100 °C). This is in contrast to the behavior of the free enzyme/proteins in the solution, which indicated sharp transitions at their respective denaturation temperatures. Denaturation of the bound protein depended on the scan rate and the denaturation process was kinetically controlled. At rapid scan rates (2 °C/min), for example, the thermal profiles of the intercalated proteins became sharper while the free proteins indicated little or no changes. Careful analysis of the calorimetric data provided a clear distinction between the moving-boundary model and the uniform distribution model for protein binding. Calorimetric data also revealed that a distribution of thermodynamic states or kinetically-slow forming states are important in the denaturation. While the thermal denaturation of Mb bound to α-ZrP indicated a significant extent of reversibility, Lys and Hb did not. The solid stabilized a fraction of the intercalated protein, and efforts will be focused to maximize this portion. Improved thermal stabilities are important for biosensor or biocatalysis applications of enzyme–inorganic materials.