Effect of sol-gel confinement on the structural dynamics of the enzyme bovine Cu,Zn superoxide dismutase.
Immobilization of proteins in sol-gel glasses has allowed the development of a new generation of robust and sensitive analytical devices as well as contributes to the investigation of the effect of molecular confinement on the structure of proteins. The immobilized protein usually preserves its structural integrity and functionality, while interactions with the matrix and its surface seem to contribute to alter its dynamics and stability. With the aim of better understanding the nature of such interactions, we have encapsulated the enzyme bovine Cu,Zn superoxide dismutase (BSOD), negatively charged at physiological pH, in a sol-gel matrix and the photophysical properties of its single tyrosine have been determined using both steady-state and time-resolved fluorescence techniques. Fluorescence spectra, quenching experiments, fluorescence lifetimes, and anisotropy measurements indicate that immobilization does not lead to any major conformational change, at least in the region of protein where the tyrosine residue is located. In addition, fluorescence anisotropy decays recorded above and below the isoelectric point of the protein indicate that, at neutral pH, well above its isoelectric point, the entrapped BSOD freely rotates within the matrix pore, but showing a different rotational behavior as compared with that in the bulk aqueous solution. However, below the isoelectric point, the global motion of the protein is totally hindered upon entrapment. Electrostatic interactions with the gel matrix, changes in water viscosity, and protein-to-pore size ratio are discussed as possible factors responsible for this behavior.
Pastor
Isabel
I
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202-Elche, Spain.
Prieto
Manuel
M
Mateo
C Reyes
CR
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000737:chemistry*
D013482:Superoxide Dismutase