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- Bohlin, Maria E, 1979-, et al.
(author)
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Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis
- 2011
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In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 32, s. 728-737
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Journal article (peer-reviewed)abstract
- 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.
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- Bohlin, Maria, et al.
(author)
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Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment
- 2012
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In: Electrophoresis. - Weinheim, Germany : John Wiley & Sons. - 0173-0835 .- 1522-2683. ; 33:12, s. 1695-1702
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Journal article (peer-reviewed)abstract
- Sample adsorption to the inner surface of fused silica capillaries is a common problem inCE when analyzingmacromolecules and is harmful to the analysis. We previously utilizedthe pH hysteresis effect of fused silica to facilitate electrophoresis of the strongly adsorbingprotein β2gpI in plain-fused silica capillaries at neutral pH. In the present paper, theeffect of different pretreatments of the capillary on the adsorption of the β2-glycoproteinI has been investigated using electroosmosis markers, SDS mobilization, and imagingbased on indirect immunofluorescence microscopy for direct visualization. The amountof β2gpI adsorbed on the surface was probed using all these independent techniques afterelectrophoresis at neutral pH on capillaries pretreated with HCl, background electrolyte(BGE), and NaOH. BGE pretreatment was included as a positive control. We found that80% or more of the starting material was adsorbed to the inner surface of the silicacapillaries during electrophoresis after pretreatment with only BGE or with NaOH, butafter acidic pretreatment the loss was consistently less than 20%. NaOH most efficientlyremoves adsorbed protein between runs. A theoretical calculation of the pH change ofthe BGE showed that electrolysis affects the pH more than the deprotonation of silanolsduring electrophoresis. We conclude that acidic pretreatment of fused silica capillariesdiminishes adsorption of β2gpI by decreasing charge-dependent wall adsorption.
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- Olsson, Jeanette, 1976-, et al.
(author)
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Enantioseparation of Omeprazole and its Metabolite 5-Hydroxyomeprazole using Open Tubular Capillary Electrochromatography with Immobilized Avidin as Chiral Selector
- 2008
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In: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 875:1, s. 329-332
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Journal article (peer-reviewed)abstract
- The present paper demonstrates the enantiomeric separation of omeprazole and its metabolite 5-hydroxyomeprazole performed with open tubular capillary electrochromatography (OT-CEC). The protein avidin was used as the chiral selector. Avidin was immobilized by a Schiffs base type of reaction where the protein was via glutaraldehyde covalently bonded to the amino-modified wall of a fused-silica capillary, 50 μm i.d. Both racemates were baseline resolved. Resolution was 1.9 and 2.3, respectively, using ammonium acetate buffer, pH 5.8, 5% methanol, with UV-detection. These values of resolution using OT-CEC are higher than earlier published results regarding chiral separation of omeprazole and 5-hydroxyomeprazole on packed CEC. The number of theoretical plates also indicated good separation efficiency.
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