Kk. Flora et Jd. Brennan, Effect of matrix aging on the behavior of human serum albumin entrapped ina tetraethyl orthosilicate-derived glass, CHEM MATER, 13(11), 2001, pp. 4170-4179
The steady-state and time-resolved fluorescence of Trp-214 was used to exam
ine the conformation, dynamics, accessibility, thermal stability, and degre
e of ligand binding of human serum albumin (HSA) after entrapment of the pr
otein in sol-gel processed glasses. The bioglasses were derived from tetrae
thyl 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, signi
ficant changes were observed in the structure and dynamics of HSA, consiste
nt with adsorption of the protein onto the silica surface combined with par
tial 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 continu
ed. All proteins showed full accessibility to neutral quenchers over 2 mont
hs of aging but only partial accessibility to negatively charged quenchers,
even at early aging times, indicating electrostatic repulsion of such anal
ytes by the negatively charged matrix. Taken together, the results indicate
d that the reduced ligand binding for entrapped HSA was caused by a combina
tion 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 entrap
ped in silicate-based glasses.