Impeded rotation of a protein in a sol-gel matrix

The sol-gel encapsulation process has been exploited in recent years for th e immobilization of proteins to be used as biosensors. Sol-gels derived fro m tetramethyl orthosilicate provide a stable environment for the macromolec ule combined with the free flow of small substrates to a protein's binding site. The functionality of a number of enzymes within the solid matrix has been demonstrated. However, very little biophysical characterization of the encapsulated proteins has been done. in this study, time-resolved fluoresc ence anisotropy was used to compare the rotational mobility of two probes i n sol-gel matrices derived from three different preparative methods. A smal l fluorescent probe, sulforhodamine 101 (SR101), was used to gauge the rela tive solvent viscosity within the sol-gels. Magnesium protoporphyrin IX sub stituted myoglobin (MgMb) provides a convenient fluorescent probe for measu ring rotational dynamics of a typical globular protein. The anisotropy deca y of the Mg-heme is sensitive only to the global protein motion. The SR101 reveals both low (phi < 1 ns) and high (phi = 6-500 ns) viscosity encapsula tion sites within the matrix, and the populations of these sites are depend ent on gel preparation and age. The protein, however, shows greatly diminis hed decay of the fluorescence anisotropy (phi similar to 1 mu s) in two of the three gels (but was denatured in the third). This is consistent with re strictive encapsulation sites where size and/or environment substantially i mpedes rotational diffusion.