Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
Karlstad University, Faculty of Technology and Science, Department of Chemistry and Biomedical Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Karlstad: Karlstad University , 2011. , 77 p.
Series
Karlstad University Studies, ISSN 1403-8099 ; 2011:48
Keyword [en]
Capillary Electrophoresis, β2-glycoprotein I, acidic pretreatment, pH hysteresis effect, Affinity Capillary Electrophoresis
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kau:diva-8277ISBN: 978-91-7063-383-6 (print)OAI: oai:DiVA.org:kau-8277DiVA: diva2:441737
Public defence
2011-10-28, Nyquistsalen, 9C 203, Karlstads Universitet, Karlstad, 10:15 (English)
Opponent
Supervisors
Note

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.

Available from: 2011-10-07 Created: 2011-09-19 Last updated: 2016-02-12Bibliographically approved
List of papers
1. Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human β2-glycoprotein I
Open this publication in new window or tab >>Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human β2-glycoprotein I
2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1059, 215-222 p.Article in journal (Refereed) Published
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-3822 (URN)10.1016/j.chroma.2004.09.087 (DOI)
Available from: 2009-03-20 Created: 2009-03-20 Last updated: 2017-12-13Bibliographically approved
2. Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of protein in bare fused-silica capillaries
Open this publication in new window or tab >>Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of protein in bare fused-silica capillaries
2005 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, no 21, 4043-4049 p.Article in journal (Refereed) Published
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-3824 (URN)10.1002/elps.200500288 (DOI)
Available from: 2009-03-20 Created: 2009-03-20 Last updated: 2017-12-13Bibliographically approved
3. Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis
Open this publication in new window or tab >>Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis
2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, 728-737 p.Article in journal (Refereed) 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. 

Keyword
affinity capillary electrophoresis; β2-glycoprotein I; conformation; heparin; pH hysteresis effect
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-8275 (URN)10.1002/elps.201000538 (DOI)000288602000012 ()
Available from: 2011-09-19 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
4. Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment
Open this publication in new window or tab >>Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment
Show others...
2012 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 12, 1695-1702 p.Article in journal (Refereed) Published
Abstract [en]

Sample adsorption to the inner surface of fused silica capillaries is a common problem in

CE when analyzingmacromolecules and is harmful to the analysis. We previously utilized

the pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbing

protein β2gpI in plain-fused silica capillaries at neutral pH. In the present paper, the

effect of different pretreatments of the capillary on the adsorption of the β2-glycoprotein

I has been investigated using electroosmosis markers, SDS mobilization, and imaging

based on indirect immunofluorescence microscopy for direct visualization. The amount

of β2gpI adsorbed on the surface was probed using all these independent techniques after

electrophoresis at neutral pH on capillaries pretreated with HCl, background electrolyte

(BGE), and NaOH. BGE pretreatment was included as a positive control. We found that

80% or more of the starting material was adsorbed to the inner surface of the silica

capillaries during electrophoresis after pretreatment with only BGE or with NaOH, but

after acidic pretreatment the loss was consistently less than 20%. NaOH most efficiently

removes adsorbed protein between runs. A theoretical calculation of the pH change of

the BGE showed that electrolysis affects the pH more than the deprotonation of silanols

during electrophoresis. We conclude that acidic pretreatment of fused silica capillaries

diminishes adsorption of β2gpI by decreasing charge-dependent wall adsorption.

 

Place, publisher, year, edition, pages
Weinheim, Germany: John Wiley & Sons, 2012
Keyword
Acidic pretreatment / Capillary electrophoresis / pH-hysteresis effect / Protein adsorption
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-30939 (URN)10.1002/elps.201100592 (DOI)000305792500003 ()22674218 (PubMedID)
Available from: 2014-01-20 Created: 2014-01-20 Last updated: 2017-08-15Bibliographically approved

Open Access in DiVA

Method development(1834 kB)551 downloads
File information
File name FULLTEXT01.pdfFile size 1834 kBChecksum SHA-512
b97da1f4f697415647b9c99714a8200f8c31f214d374213ef1c69d75910c3f6e7c6ec4a744cce71e0e3e4affeb583467c3c9074d585a3e6f696cf60c288d85eb
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Bohlin, Maria E.
By organisation
Department of Chemistry and Biomedical Sciences
Analytical Chemistry

Search outside of DiVA

GoogleGoogle Scholar
Total: 551 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 340 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf