Affinity-CE with phospholipid and carbohydrate ligands : Binding between heparin and anionic liposomes and the anticoagulant protein, beta-2-glycoprotein I

• Binding studies executed by capillary electrophoresis (CE) benefit from flexibility in experimental set-up, wide applicability, and low consumption of reagents. Importantly, the high resolution inherent in CE allows complex mixtures and binding resulting in only small migration changes to be characterized quantitatively. One specific problem, however, that may be encountered when applying CE for the analysis of binding interactions of proteins is the need to avoid interactions other than those between the analyte and the ligand. Thus, protein-wall interactions in unmodified fused-silica capillaries used at neutral pH often invalidate analyses of protein binding. This problem is especially pronounced with basic proteins and proteins containing exposed patches of positive charge. Ways to overcome this are to neutralize the immobilized wall charges e.g. by different wall coatings and/or buffer additives, or by using the pH hysteresis effect, i.e., the slow deprotonation at neutral pH of low pH-treated fused silica. We illustrate the use of the pH-hysteresis effect to avoid adsorption problems in the analysis of a phospholipid- and heparin-binding anticoagulant protein, beta-2-glycoprotein under physiological pH conditions. The approach was useful for characterizing protein-heparin and protein-liposome binding. This will be the basis of using CE methods to evaluate the influence of human thrombogenic autoantibodies against this protein on such interactions and the approach will be also be a useful means to measure the binding activity of different domains and structurally modified variants of this protein and other proteins that suffer from a tendency to stick to fused silica

Subject headings

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Keyword

Chemistry

Kemi

Publication and Content Type

pop (subject category)

kon (subject category)