218 Langmuir 1991, 7, 218-222 Articles Fluorescence Quenching of Adsorbed Hen and Human Lysozymes D. Horsley, J. Herron, V. Hlady,+and J. D. Andrade* Center for Biopolymers at Interfaces, Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112 Received December 5, 1989. I n Final Form: July 2, 1990 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 experimental 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. Introduction The understanding and control of the interactions of proteins with solid surfaces are important in a number of areas of biology and medicine. In the last 20 years, there has been considerable interest in protein interactions with materials used in medical implant devices.'-3 One area of particular interest to the contact lens industry is the interaction of tear proteins with contact l e n ~ e s . ~ - ~ Dep- osition of proteins on contact lens surfaces cause a loss of visual acuity (due to the opaque nature of the adsorbed protein film), wearer discomfort, and, in some cases, acute eye disease~.~J~ Lysozyme is a major protein constituent in tear fluids and has been shown to also be a major * To whom correspondence should be addressed. t On the leave of absence from "Ruder BoSkoviE" Institute, Za- greb, Yugoslavia. (1) Vroman, L., Leonard, E. F., Eds. Ann. N.Y. Acad. Sci. 1977,283. (2) Baier, R. E., Ed. Applied Chemistry at Protein Interfaces. Ado. Chem. Ser. 1975, No. 145. (3) Cooper, S. L., Peppas, N. A., Eds. Biomaterials: Interfacial Phenomena and Applications. Ado. Chem. Ser. 1982, No. 199. (4) Karageozian, H. L. Chemical Identity of Opaque Deposits on Human Worn Hydrophilic Lenses; Report Series 92; Allergan Pharma- ceuticals: Irvine, CA, 1974. (5) Wedler, F. C. J. Biomed. Mater. Res. 1977, 11, 525. (6) Wedler, F. C.; Riedhammer, T. M. In CRC Critical Reoiews, Bio- compatibility; Williams, D. F., Ed.; CRC Press: Cleveland, OH, 1979. (7) Hosaka, S.; Ozawa, H.; Tanzawa, H.; Ishida, H.; Yoshimura, K.; Momose, T.; Nakajima, A. J. Biomed. Mater. Res. 1983,17,261. (8) Tripathi, R.; Montague, R.; Tripathi, B. J. In Soft Contact Lenses; Clinical and Applied Technology; Ruben, M., Ed.; J. Wiley and Sons: New York, 1978; p 299. (9) Allansmith, M. R.; Korb, D. R.; Greiner, J. V.; Henriquez, A. S.; Simon, M. A.; Finnemore, V. M. Am. J. Ophthalmol. 1977,83,697. (10) Dohlman, C. H.; Boruchoff, S. A.; Mobilia, E. F. Arch. Ophthal- mol. 1973, 90, 367. 0743-7463/91/2407-0218$02.50/0 component in soft contact lens dep~sits.ll-'~ Conse- quently, the use of lysozyme in protein adsorption studies has great practical value. Furthermore, since lysozyme is a rather simple, well-understood p r ~ t e i n , ' ~ J ~ it can easily serve as a model for understanding the general principles that seem to govern protein adsorption. Experimental methods used to study the structural changes that occur in proteins as they undergo adsorption at interfaces are currently very limited. Total internal reflection intrinsic fluorescence (TIRIF) spectroscopy combines well-known advantages of fluorescence spec- troscopy with the surface sensitivity of internal reflection optics and it has been used in different aspects of protein adsorption.lelg One of the fluorescence techniques that is often used to probe protein conformational changes in solution is the technique of fluorescence q~enching.~O-~~ Presently, very little is done with this technique to probe (11) Bilbaut, T.; Gachon, A. M.; Dastugue, B. Exp. Eye Res. 1986,43, 153. (12) Gachon, A. M.; Bilbaut, T.; Dastugue, B. Exp. Eye Res. 1985,43, 105. (13) Royce, F. H., Jr.; Ratner, B. D.; Horbett, T. A. Ado. Chem. Ser. 1982, No. 199,453. (14) Imoto, T.; Johnson, L. N.; North,A. C. T.; Phillips, D. C.; Rupley, J. A. In The Enzymes; Boyer, P. D., Ed., Academic Press: New York, 1972; Vol 15, p 665. (15) Osserman, E. F., Canfield, R. E., Beychok, S., Eds. Lysozyme; Academic Press: New York, 1974. 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