Concomitant isolation of protein C inhibitor and unnicked β2-glycoprotein I

beta(2)-Glycoprotein I (beta(2)GPI) is the major target molecule for socalled anticardiolipin antibodies. We evaluated the isolation procedure of beta(2)GPI from human plasma with special emphasis on the time of precipitation, composition of different isolated fractions and their antigenic properties. The isolation was initiated by perchloric acid precipitation for either 3, 18 or 50 min, followed by heparin affinity and cationic exchange chromatography. The properties of isolated proteins were tested by rocket electrophoresis, enzymelinked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and Nterminal sequencing. Each isolation procedure, regardless of the perchloric acid precipitation duration, resulted in three distinct protein peaks, differing in protein composition qualitatively. Comparing sequential peaks between the isolations of different precipitation times, we found that all the three first peaks (set of peaks No. 1), all the three second peaks (set No. 2) as well as the three third peaks (set No. 3) consisted of identical proteins but in different quantities. Set No. 1 was composed of immunoglobulins and a lesser amount of beta(2)GPI. In set No. 2 only unnicked beta(2)GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked beta(2)GPI in set No. 3. Oxidation or degradation of beta(2)GPI during the isolation procedure did not result in a mixture of different forms of beta(2)GPI but rather in a lower yield of wildtype beta(2)GPI. The coexistence of beta(2)GPI and protein C inhibitor in the isolated fractions may suggest their proteinprotein interactions in vivo.