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Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis
Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
Karlstads universitet, Fakulteten för teknik- och naturvetenskap, Avdelningen för kemi och biomedicinsk vetenskap.
Statens Serum Institut, Copenhagen.
2011 (engelsk)Inngår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, s. 728-737Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

We have used CE to evaluate the interaction between β2-glycoprotein I (β2gpI) and heparin. β2gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of β2gpI because the exact function is not entirely known and because circulating autoantibodies against β2gpI are associated with an increased risk of thrombotic events.

 

The effect of the ionic strength, temperature, and conformation of the protein on the interaction between β2gpI and heparin has been studied. The CE procedure for this study is simple, fast and automatic. β2gpI and heparin were allowed to interact during electrophoresis at different ionic strength buffers and at different capillary temperatures. To mimic perturbation of the conformation of β2gpI, different denaturing agents (SDS, ACN and urea) were added to the background electrolyte. While simple 1:1 binding isotherms were obtained at 22 °C the data strongly suggests that at physiological temperature the binding stoichiometry is not 1:1 and/or that cooperative interactions begin to play a role. We found that (i) the KD values differed by a factor of 60 at the ionic strengths studied (ii) β2gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea and (iii) the KD for the β2gpI-heparin interaction in the presence of urea was 10 times higher than the KD determined at the same conditions without urea added. Therefore, we conclude that the interaction between β2gpI and heparin is dependent on electrostatic interactions and on the conformation of β2gpI. 

sted, utgiver, år, opplag, sider
2011. Vol. 32, s. 728-737
Emneord [en]
affinity capillary electrophoresis; β2-glycoprotein I; conformation; heparin; pH hysteresis effect
HSV kategori
Forskningsprogram
Kemi
Identifikatorer
URN: urn:nbn:se:kau:diva-8275DOI: 10.1002/elps.201000538ISI: 000288602000012OAI: oai:DiVA.org:kau-8275DiVA, id: diva2:441725
Tilgjengelig fra: 2011-09-19 Laget: 2011-09-19 Sist oppdatert: 2018-01-15bibliografisk kontrollert
Inngår i avhandling
1. Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
Åpne denne publikasjonen i ny fane eller vindu >>Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
2011 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The final goal of this study is to establish a microscale analysis method that allows solution phase characterization of interactions between β2-glycoprotein I (β2gpI) and some of its ligands. Human β2gpI is a phospholipid- and heparin-binding plasma glycoprotein. The physiological role of the protein in normal blood coagulation is not entirely known, nor is its role in autoimmune diseases characterized by blood clotting disturbances (thrombosis). Quantitative binding data of β2gpI interactions with some of its ligands may help elucidating the mechanisms behind these diseases and in the development of new approaches for diagnostics, prevention, and therapy.

In this thesis, capillary electrophoresis (CE) was used as methodological platform for the interaction studies. The analysis of peptides and proteins by CE is desirable due to low sample consumption, possibilities for non-denaturing and highly effective separations. The first objective of this thesis was to find an approach to prevent charge dependent adsorption of β2gpI to the inner surface of the capillaries. Analyte adsorption at the negatively charged inner surface of fused silica capillaries is detrimental to interaction analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged domains, such as β2gpI. A new strategy to suppress these solute-wall interactions was devised, investigated and optimized. This strategy exploits the pH hysteresis behavior of fused silica surfaces, by simply performing an acidic pretreatment of the capillary. The results in this thesis show that the acidic pretreatment efficiently prevents protein adsorption.

sted, utgiver, år, opplag, sider
Karlstad: Karlstad University, 2011. s. 77
Serie
Karlstad University Studies, ISSN 1403-8099 ; 2011:48
Emneord
Capillary Electrophoresis, β2-glycoprotein I, acidic pretreatment, pH hysteresis effect, Affinity Capillary Electrophoresis
HSV kategori
Forskningsprogram
Kemi
Identifikatorer
urn:nbn:se:kau:diva-8277 (URN)978-91-7063-383-6 (ISBN)
Disputas
2011-10-28, Nyquistsalen, 9C 203, Karlstads Universitet, Karlstad, 10:15 (engelsk)
Opponent
Veileder
Merknad

Papper 4 Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment ingick som manuskript i avhandlingen, nu publicerad.

Tilgjengelig fra: 2011-10-07 Laget: 2011-09-19 Sist oppdatert: 2016-02-12bibliografisk kontrollert

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