Effect of Matrix Aging on the Behavior of Human Serum Albumin Entrapped in a Tetraethyl Orthosilicate-Derived Glass

The steady-state and time-resolved fluorescence of Trp-214 was used to examine the conformation, dynamics, accessibility, thermal stability, and degree of ligand binding of human serum albumin (HSA) after entrapment of the protein in sol−gel processed glasses. The bioglasses were derived from tetraethyl orthosilicate and were aged in air without washing (dry-aged), in air after a washing step (washed), or in buffer (wet-aged). In all cases, significant changes were observed in the structure and dynamics of HSA, consistent with adsorption of the protein onto the silica surface combined with partial unfolding of the protein. Significant changes in the thermal stability and degree of ligand binding of the entrapped protein were also observed, with both stability and ligand binding capacity decreasing as aging continued. All proteins showed full accessibility to neutral quenchers over 2 months of aging but only partial accessibility to negatively charged quenchers, even at early aging times, indicating electrostatic repulsion of such analytes by the negatively charged matrix. Taken together, the results indicated that the reduced ligand binding for entrapped HSA was caused by a combination of protein denaturation and partial inaccessibility of the protein to negatively charged species. After 2 months of aging the entrapped proteins retained less than 15% of their binding ability in solution, regardless of which method was used to age the material. In light of these results, it is clear that improved sol−gel processing methods will be needed to overcome the time-dependent changes in the structure and function of proteins entrapped in silicate-based glasses.