Understanding the details of enzyme behavior at solid surfaces is essential for the rational design of synthetic materials for the design of biosensors, implants, enzyme reactors, and biomedical devices. To make progress toward this goal, we chose to study enzyme behavior at an ultra thin film of inorganic materials such as α-Zr(HPO₄).H₂O (abbreviated as α-ZrP) and α-Zr(O₃PCH₂CH₂COOH)₂.xH₂O (abbreviated as α-ZrCEP). The inorganic materials were adsorbed onto a monolayer of protein prepared on a Au substrate.  This set-up allowed us to self-assemble the inorganic material on the Au, and monitor the behavior of the adsorbed protein, in real time, by surface plasmon resonance spectroscopy (SPR, Chart below). The protein binding, and the urea denaturation/refolding of the adsorbed protein are monitored by SPR in real time.  The ΔG⁰ for the denaturation of cytochrome c, for example, has been estimated from the SPR studies and these indicate that the protein bound to the Au surface is less stable than in solution, but the adsorption of the inorganic layer enhanced its stability by more than 1 kcal/mol.  Our results suggest the strong role of interactions between the solid and the protein in controlling the behavior of the bound protein. These are the very first studies to directly measure protein-inorganic interactions, and these provide some of the first attempts in directly monitoring protein stability at well-defined solid surfaces.

Chart: Proteins embedded between the inorganic layer and the Au film.