Aggregation of lysozyme and of poly(ethylene glycol)-modified lysozyme after adsorption to silica.
Surface-induced aggregation is a common instability during protein storage, delivery and purification. This aggregation can lead to the formation of fibrils rich in intermolecular beta-sheet structure. Techniques to probe surface-clustering are limited. Here we use protein intrinsic fluorescence and thioflavin T probe fluorescence in a total internal reflection fluorescence (TIRF) sampling geometry to simultaneously monitor the kinetics of adsorption and aggregation for chicken egg lysozyme on a silica surface. We observe a slow surface-induced aggregation process that continues well after the lysozyme adsorption kinetics have plateaued. The rate of surface-induced aggregation is independent of the lysozyme concentration in solution. Consistent with the clustering observed via thioflavin T fluorescence, infrared amide I band spectra also show a 1.5-fold increase in intermolecular beta-sheet content upon lysozyme adsorption. Tryptophan emission spectra show no evidence for any tertiary structural change upon adsorption. Furthermore, we observe that the covalent modification of lysozyme with a single poly(ethylene glycol) (PEG) grafted chain does not inhibit aggregation on the surface, but a second PEG graft significantly inhibits the intermolecular beta-sheet formation.
Daly
Susan M
SM
Department of Chemical Engineering and Department of Biomedical Engineering, Center for Complex Fluids Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Przybycien
Todd M
TM
Tilton
Robert D
RD
D000327:Adsorption; D052160:Benzothiazoles; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012822:Silicon Dioxide; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T; D011092:Polyethylene Glycols; D012822:Silicon Dioxide; D014364:Tryptophan; D009113:Muramidase