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Capillary electrophoresis-based analysis of phospholipid- and glycosaminoglycan-binding by human beta-2-glycoprotein I
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
2004 (English)In: / [ed] E. Kogutowska, L. Blomberg, N. H. H. Heegaard, 2004Conference paper, (Other (popular science, discussion, etc.))
Abstract [en]

Human beta-2-glycoprotein I (b2gpI) is a phospholipid- and heparin-binding plasma glycoprotein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against b2gpI. With the final goal of assessing autoantibody influence on binding interactions of b2gpI we have studied the development of capillary electrophoresis (CE)-based assays for interactions of negatively charged ligands with b2gpI. Binding studies executed by capillary electrophoresis benefit from flexibility in experimental set-up, wide applicability, and low consumption of reagents.

Importantly, the high resolution inherent in CE allows complex mixtures and binding resulting in only small migration changes to be characterized quantitatively. One specific problem, however, that may be encountered when applying CE for the analysis of binding interactions of proteins is the need to avoid interactions other than those between the analyte and the ligand. Thus, protein-wall interactions in unmodified fused-silica capillaries used at neutral pH often invalidate analyses of protein binding. This problem is especially pronounced with basic proteins and proteins containing exposed patches of positive charge. In the development of suitable conditions for analysis at neutral pH of b2gpI we found the pH hysteresis behavior of fused silica surfaces useful since the protonated surface after an acid pre-wash counteracted protein adsorption efficiently in contrast to more laborious procedures including acrylamide/dimethylacrylamide coatings that did not permit analysis of this particular protein. This approach made quantitative studies of heparin-b2gpI interactions possible and the principle was shown also to work for detection of b2gpI binding to anionic phospholipids. Utilizing the pH hysteresis effect may be a simple solution to the adsorption problems often encountered in analyses of proteins by CE.

Place, publisher, year, edition, pages
2004.
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kau:diva-17570OAI: oai:DiVA.org:kau-17570DiVA: diva2:591178
Conference
IOPMS 2004, Riva del Garda, Italien
Available from: 2013-01-21 Created: 2013-01-21 Last updated: 2013-01-21

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