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Bohlin, Maria E.
Alternative names
Publications (10 of 16) Show all publications
Bohlin, M., Johannesson, I., Carlsson, G., Heegaard, N. H. & Blomberg, L. G. (2012). Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment. Electrophoresis, 33(12), 1695-1702
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
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2012 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 12, p. 1695-1702Article 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
Keywords
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: 2019-09-19Bibliographically approved
Bohlin, M. E., Blomberg, L. G. & Heegaard, N. H. (2011). Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis. Electrophoresis, 32, 728-737
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, p. 728-737Article 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. 

Keywords
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: 2018-01-15Bibliographically approved
Bohlin, M. E. (2011). Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands. (Doctoral dissertation). Karlstad: Karlstad University
Open this publication in new window or tab >>Method development for affinity capillary electrophoresis of ß2-glycoprotein I and biological ligands
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. p. 77
Series
Karlstad University Studies, ISSN 1403-8099 ; 2011:48
Keywords
Capillary Electrophoresis, β2-glycoprotein I, acidic pretreatment, pH hysteresis effect, Affinity Capillary Electrophoresis
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-8277 (URN)978-91-7063-383-6 (ISBN)
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
Bohlin, M., Blomberg, L. G., Olsson, O. & Heegaard, N. H. . (2011). Structure-activity studies of human beta2-glycoprotein I using capillary electrophoresis. Paper presented at Joint congress 2011: Microscale Bioseparations San Diego. Paper presented at Joint congress 2011: Microscale Bioseparations San Diego.
Open this publication in new window or tab >>Structure-activity studies of human beta2-glycoprotein I using capillary electrophoresis
2011 (English)Conference paper, Published paper (Refereed)
Abstract

We have investigated various modes of CE to evaluate the interaction between beta2-glycoprotein I (b2gpI) and a number of anionic ligands to contribute to the elucidation of the structure-function relationship of b2gpI. b2gpI is a plasma protein which is involved in the blood coagulation cascade under normal, physiological conditions, however, its precise function is undefined. It is also involved in pathological conditions such as the so-called anti-phospholipid syndrome, where anti-b2gpI autoantibodies induce a prothrombotic state. Therefore, functional characterization of b2gpI under near physiological conditions is of interest.

To avoid charge-dependent analyte adsorption to the inner surface of the capillary wall, we have utilized the pH hysteresis effect, where an acidic pretreatment of the capillary made it possible to perform subsequent CE analyses of b2gpI at neutral pH.

The interaction between b2gpI and the anionic ligand heparin was studied with migration shift ACE, where the ionic strength, temperature and conformation of b2gpI were easily varied. The interaction between b2gpI and phosphatidylcholine/phosphatidylserine liposomes are subject to an ongoing investigation by means of migration shift ACE, frontal analysis CE, partial filling CE and pre-equilibration partial filling ACE.

We conclude that differential, but relatively low binding affinities that are highly dependent on electrostatic interactions and on a preserved conformation of the protein, characterize its interactions with ligands that in vivo will be present in multiple copies on e.g. cell surfaces. The CE procedure for this study is simple, fast and automatic and quantitative binding affinity parameters are conveniently obtained using small amounts of biological materials.

National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-10742 (URN)
Conference
Joint congress 2011: Microscale Bioseparations San Diego
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2013-06-12Bibliographically approved
Bohlin, M. & Blomberg, L. G. (2010). Affinity studies of beta-2-glycoprotein I using capillary electrophoresis. Paper presented at Microscale Bioseparations 2010, Prag, Tjeckien. Paper presented at Microscale Bioseparations 2010, Prag, Tjeckien.
Open this publication in new window or tab >>Affinity studies of beta-2-glycoprotein I using capillary electrophoresis
2010 (English)In: / [ed] Lars G. Blomberg, Niels H. H. Heegaard, 2010Conference paper, Published paper (Refereed)
Abstract

Beta2-glycoprotein I (b2gpI), also known as apolipoprotein H, is a plasma protein which is involved in the blood coagulation cascade. It binds negatively charged substances such as heparin, DNA, and anionic phospholipids. A number of functions of b2gpI have been proposed, however, the precise function is still not entirely known. Circulating autoantibodies against b2gpI are associated with increased risk of thrombotic events, such as thrombosis and reoccurring fetal loss. It is therefore of interest to functionally characterize b2gpI including the influence of anti-b2gpI autoantibodies on the ligand binding behavior of the protein. The characterization of interactions between biological molecules may be accomplished by capillary electrophoresis under non-denaturing conditions, without the need for immobilization. To avoid charge dependent analyte adsorption to the negative charges of the capillary wall we found the pH hysteresis effect of silica very useful. An acidic pretreatment of the capillary made it possible to perform a subsequent analysis at neutral pH. We were able to perform binding studies between b2gpI and heparin and monosaccharides at different ionic strengths and temperatures in a simple way. We could also study the effect of mildly denaturing conditions on the binding to the different ligands simply by adding sodium dodecyl sulfate (SDS), urea and ACN to the background electrolyte.

The approach is simple, fast and automatic. The ionic strength, temperature and other parameters such as denaturing agents could easily be changed to characterize the binding between b2gpI and different ligands.

National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-10136 (URN)
Conference
Microscale Bioseparations 2010, Prag, Tjeckien
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2013-06-12Bibliographically approved
Bohlin, M. & Blomberg, L. G. (2010). Affinity studies of beta2-glycoprotein I using capillary electrophoresis. Paper presented at Analysdagarna 2010, Uppsala. Paper presented at Analysdagarna 2010, Uppsala.
Open this publication in new window or tab >>Affinity studies of beta2-glycoprotein I using capillary electrophoresis
2010 (English)In: / [ed] Lars G. Blomberg, Niels Heegaard, 2010Conference paper, Published paper (Refereed)
Abstract

beta2-glycoprotein I (b2gpI), also known as apolipoprotein H, is a plasma protein which is involved in the blood coagulation cascade. It binds negatively charged substances such as heparin, DNA, and anionic phospholipids. A number of functions of b2gpI have been proposed, however, the precise function is still not entirely known. Circulating autoantibodies against b2gpI are associated with increased risk of thrombotic events, such as thrombosis and reoccurring fetal loss. It is therefore of interest to functionally characterize b2gpI including the influence of anti-b2gpI autoantibodies on the ligand binding behavior of the protein. The characterization of interactions between biological molecules may be accomplished by capillary electrophoresis under non-denaturing conditions, without the need for immobilization. To avoid charge dependent analyte adsorption to the negative charges of the capillary wall we found the pH hysteresis effect of silica very useful. An acidic pretreatment of the capillary made it possible to perform a subsequent analysis at neutral pH. We were able to perform binding studies between b2gpI and heparin at different ionic strengths and temperatures in a simple way. We could also study the effect of mildly denaturing conditions on the binding to the ligand simply by adding sodium dodecyl sulfate (SDS), urea and ACN to the background electrolyte. The approach is simple, fast and automatic

National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-10137 (URN)
Conference
Analysdagarna 2010, Uppsala
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2013-06-12Bibliographically approved
Bohlin, M. (2009). Capillary electrophoresis of b2-glycoprotein I: Method Development for Binding Studies. Paper presented at Current Aspects of Analytical Chemistry, Karlstad. Paper presented at Current Aspects of Analytical Chemistry, Karlstad.
Open this publication in new window or tab >>Capillary electrophoresis of b2-glycoprotein I: Method Development for Binding Studies
2009 (English)In: / [ed] Lars G. Blomberg, Niels H. H. Heegaard, 2009Conference paper, Published paper (Other (popular science, discussion, etc.))
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-10138 (URN)
Conference
Current Aspects of Analytical Chemistry, Karlstad
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2013-06-12Bibliographically approved
Bohlin, M. (2006). Capillary electrophoresis of beta2-glycoprotein I: Method development and binding studies. (Licentiate dissertation). Karlstad
Open this publication in new window or tab >>Capillary electrophoresis of beta2-glycoprotein I: Method development and binding studies
2006 (English)Licentiate thesis, monograph (Other academic)
Place, publisher, year, edition, pages
Karlstad: , 2006
Series
Karlstad University studies, 1403-8099 ; 2006:43
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-17568 (URN)9170630747 (ISBN)
Available from: 2013-01-21 Created: 2013-01-21 Last updated: 2013-01-21
Bohlin, M. E., Blomberg, L. G. & Heegard, N. H. .. (2005). Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of protein in bare fused-silica capillaries. Electrophoresis, 26(21), 4043-4049
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, p. 4043-4049Article 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
Bohlin, M., Blomberg, L. G. & Heegaard, N. (2005). Utilizing the pH Hysteresis Effect for Versatile and Simple Electrophoretic Analysis of Proteins in Bare Fused-Silica Capillaries. Electrophoresis, 26 (2005) 4043-4049
Open this publication in new window or tab >>Utilizing the pH Hysteresis Effect for Versatile and Simple Electrophoretic Analysis of Proteins in Bare Fused-Silica Capillaries
2005 (English)In: Electrophoresis, 26 (2005) 4043-4049Article in journal (Refereed)
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-25317 (URN)
Available from: 2013-01-22 Created: 2013-01-22 Last updated: 2013-01-22
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