FLUORESCENCE QUENCHING OF ADSORBED HEN AND HUMAN LYSOZYMES

The iodide quenching of protein fluorescence was used to study the effect of surface adsorption on the conformation of human and hen lysozymes. Three different types of surfaces were used for protein adsorption: (a) hydrophobicized, uncharged DDS-silica; (b) unmodified, negatively charged silica; (c) positively charged APS-silica. The evanescent surface wave generated by total internal reflection was used to excite intrinsic fluorescence from the tryptophanyl residues of irreversibly adsorbed lysozymes. The extent of quenching of the adsorbed lysozyme fluorescence was shown to be a function of both the species of lysozyme studied (human vs hen) and the type of surface to which the protein was adsorbed. A modified Stern-Volmer quenching model, which assumes accessible and inaccessible populations of protein fluorophores, was applied to analyze the expeimental results. The change in the fractional accessibility of fluorophores due to the adsorption was taken as a measure of protein conformational change. Both lysozymes appeared to exhibit smaller denaturation at the DDS-silica surface than on the other two surfaces. According to the quenching results both lysozymes were at least partially denatured upon adsorption to the unmodified, negatively charged silica surface as well as on the positively charged APS-silica surface. Human lysozyme displayed much larger changes in the denaturation parameters upon adsorption to the three surfaces than the hen lysozyme, indicating that it is less conformationally stable at interfaces. It was found that the effective quenching constant of iodide anion depended largely on the charge of the surface.