We have used CE to evaluate the interaction between beta(2)-glycoprotein I (beta(2)gpI) and heparin. beta(2)gpI is a human plasma protein involved in the blood coagulation cascade. It is of interest to functionally characterize the interactions of beta(2)gpI because the exact function is not entirely known and because circulating autoantibodies against beta(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 beta(2)gpI and heparin has been studied. The CE procedure for this study is simple, fast, and automatic. beta(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 beta(2)gpI, different denaturing agents (SDS, ACN, and urea) were added to the BGE. While simple 1:1 binding isotherms were obtained at 22 degrees 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) beta(2)gpI was resistant to denaturation with SDS and ACN, but was partially denatured by urea, and (iii) the K-D for the beta(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 beta(2)gpI and heparin is dependent on electrostatic interactions and on the conformation of beta(2)gpI.