title: Effects of ionic strength, temperature and conformation on affinity interactions of β₂-glycoprotein I monitored by capillary electrophoresis.
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 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) β₂gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea, and (iii) the K(D) for the β₂gpI-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 β₂gpI and heparin is dependent on electrostatic interactions and on the conformation of β₂gpI.
authors.affiliation: Department of Chemistry and Biomedical Sciences, Karlstad University, Karlstad, Sweden. maria.bohlin@kau.se
authors.forename: Maria E
authors.initials: ME
authors.lastname: Bohlin
authors.forename: Lars G
authors.initials: LG
authors.lastname: Blomberg
authors.forename: Niels H H
authors.initials: NH
authors.lastname: Heegaard
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D058767:Protein Unfolding; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013696:Temperature; D014508:Urea / Q000737:chemistry; D053482:beta 2-Glycoprotein I / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000097:Acetonitriles; D053482:beta 2-Glycoprotein I; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D006493:Heparin; C032159:acetonitrile