Maria Bohlin

Separation of conjugated trienoic fatty acid isomers by capillary electrophoresis.

A method for direct resolution of conjugated trienoic fatty acid isomers by capillary electrophoresis has been developed. To obtain complete separation a dual cyclodextrin system was used. This contained heptakis-(6-sulfo)-beta-cyclodextrin (charged). Beta-cyclodextrin (uncharged) and sodium dodecylsulfate. Under optimized conditions, all seven isomers were well separated. On average, separation efficiency was 2.9 x 10(5) plates/m.

Effects of ionic strength, temperature and conformation on affinity interactions of β2-glycoprotein I monitored by capillary electrophoresis

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 BGE. While simple 1:1 binding isotherms were obtained at 22°C, the data strongly suggest 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 ten 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.

Estimation of the amount of β2-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment

Sample adsorption to the inner surface of fused silica capillaries is a common problem in CE when analyzing macromolecules 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.