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Thermodynamic Analysis of Proteins Adsorbed on Silica Particles: Electrostatic Effects
Uppsala University, Centre for Surface Biotechnology.
2001 In: Journal of Colloid and Interface Science, Vol. 237, no 1, 98-103 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2001. Vol. 237, no 1, 98-103 p.
URN: urn:nbn:se:uu:diva-92363OAI: oai:DiVA.org:uu-92363DiVA: diva2:165409
Available from: 2004-11-17 Created: 2004-11-17Bibliographically approved
In thesis
1. Macromolecules at Interfaces
Open this publication in new window or tab >>Macromolecules at Interfaces
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Makromolekyler på ytor
Abstract [en]

In this thesis, the structure and stability of globular proteins adsorbed onto nanometer-sized hydrophilic silica particles were investigated using differential scanning calorimetry (DSC), hydrogen/deuterium exchange (HDX), and mass spectrometry (MS). The adsorption process itself was characterized with fluorescence and absorption spectroscopy and surface plasmon resonance (SPR). The combination of these methods offered a unique insight into adsorption-induced changes within proteins related to their adsorption characteristics. DSC contributed with thermodynamic information on the overall structural stability within the protein population. HDX in combination with MS contributed information on the structure and stability of adsorbed proteins with focus on changes within the secondary structure elements. In order to increase the structural resolution in this part of the investigation, proteolysis was performed prior to the MS analyzing step. Knowledge on the protein adsorption process was utilized in a practical approach called ligand fishing. In this approach, SPR was used to monitor the chip-based affinity purification of a protein with MS used for protein identification.

Adsorption isotherms revealed that electrostatic interactions play an important role in the adsorption of proteins to hydrophilic surfaces. DSC investigation revealed that the thermal stability of proteins reduces with increasing electrostatic attraction between the protein and the surface and that this effect diminishes at higher surface coverage. The mass-increase due to exchange between protein hydrogen atoms and deuterium atoms in solution was investigated as a function of time. This gave insight into adsorption-induced changes in the structural stability of proteins. By combining DSC and HDX-MS, it was possible to differentiate between adsorption-induced changes in the secondary and tertiary structure. Additionally, if limited proteolysis was performed, the investigations gave insight into the orientation and protein segment specific changes in the stability of proteins adsorbed to silica surfaces. The adsorption of proteins to silica particles also provided the basis for a new experimental design that allows handling of minute amounts of proteins in a ligand fishing application, as used in the field of functional proteomics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 64 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 1039
Chemistry, Protein, Adsorption, Immobilization, Surface, Surface Plasmon Resonance, SPR, Mass Spectrometry, MS, Differential Scanning Calorimetry, DSC, FT ICR, Lysozyme, Bovine Serum Albumin, BSA, Myoglobin, Globular, Kemi
National Category
Chemical Sciences
urn:nbn:se:uu:diva-4661 (URN)91-554-6089-5 (ISBN)
Public defence
2004-12-09, C10:301, BMC, Husargatan 3, Uppsala, 13:15
Available from: 2004-11-17 Created: 2004-11-17Bibliographically approved

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