Effects of ionic strength, temperature and conformation on affinity interactions of β 2 ‐glycoprotein I monitored by capillary electrophoresis

We have used CE to evaluate the interaction between β 2 ‐glycoprotein I (β 2 gpI) and heparin. β 2 gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of β 2 gpI because the exact function is not entirely known and because circulating autoantibodies against β 2 gpI 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 β 2 gpI and heparin has been studied. The CE procedure for this study is simple, fast, and automatic. β 2 gpI 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 β 2 gpI, 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) β 2 gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea, and (iii) the K D for the β 2 gpI–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 β 2 gpI and heparin is dependent on electrostatic interactions and on the conformation of β 2 gpI.