Estimation of the amount of ß2-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 ß(2) gpI 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 ß(2) -glycoprotein I has been investigated using electroosmosis markers, SDS mobilization, and imaging based on indirect immunofluorescence microscopy for direct visualization. The amount of ß(2) gpI 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 ß(2) gpI by decreasing charge-dependent wall adsorption.

Effects of ionic strength, temperature and conformation on affinity interactions of ß2-glycoprotein I monitored by capillary electrophoresis.

We have used CE to evaluate the interaction between ß2-glycoprotein I (ß2gpI) and heparin. ß2gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of ß2gpI because the exact function is not entirely known and because circulating autoantibodies against ß2gpI 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 ß2gpI and heparin has been studied. The CE procedure for this study is simple, fast, and automatic. ß2gpI 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 ß2gpI, 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 K(D)-values differed by a factor of 60 at the ionic strengths studied (ii) ß2gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea, and (iii) the K(D) for the ß2gpI-heparin interaction in the presence of urea was ten times higher than the K(D) determined at the same conditions without urea added. Therefore, we conclude that the interaction between ß2gpI and heparin is dependent on electrostatic interactions and on the conformation of ß2gpI.

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.

Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human beta2-glycoprotein I.

Human beta2-glycoprotein I (beta2gpI) is a phospholipid and heparin binding plasma glycoprotein involved in autoimmune diseases characterized by blood clotting disturbances (thrombosis) together with the occurrence of autoantibodies against beta2gpI. With the final goal of assessing autoantibody influence on binding interactions of beta2gpI we have studied the development of capillary electrophoresis (CE)-based assays for interactions of negatively charged ligands with beta2gpI. In the development of suitable conditions for analysis at neutral pH of this basic protein (pI about 8) we found the pH hysteresis behavior of fused silica surfaces useful since the protonated surface after an acid pre-wash counteracted protein adsorption efficiently in contrast to more laborious procedures including acrylamide/dimethylacrylamide coatings that did not permit analysis of this particular protein. This simple approach made estimates of heparin-beta2gpI interactions possible and the principle was shown also to work for detection of betagpI binding to anionic phospholipids. Utilizing the pH hysteresis effect may be a simple solution to the adsorption problems often encountered in analyses of proteins by CE.

Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries.

The analysis of peptides and proteins by CE is often desirable due to low sample consumption and possibilities for nondenaturing yet highly effective separations. However, adsorption to the inner surfaces of fused-silica capillaries often is detrimental to such analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged patches. To avoid wall interactions numerous buffer additives and static and dynamic wall coating principles have been devised. We previously showed (J. Chromatogr. A 2004, 1059, 215-222) that CE of the basic protein beta2-glycoprotein was rendered possible by an acidic pretreatment step, and we attributed this observation to the so-called pH hysteresis effect that influences the time for pH equilibration of the capillary wall and thus the effective wall charge and the electroosmotic mobility. We here investigate the effects of different pretreatment techniques on EOF values and on the rate of the deprotonation of silanol groups when performing the electrophoresis at neutral pH. We show the utility of this simple approach for the CE analysis of a number of basic proteins in plain silica capillaries at physiological pH.