Concomitant isolation of protein C inhibitor and unnicked beta2-glycoprotein I.
beta2-Glycoprotein I (beta2GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of beta2GPI 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, enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and N-terminal 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 beta2GPI. In set No. 2 only unnicked beta2GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked betaGPI in set No. 3. Oxidation or degradation of beta2GPI during the isolation procedure did not result in a mixture of different forms of beta2GPI but rather in a lower yield of wild-type beta2GPI. The co-existence of beta2GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.
Cucnik
Sasa
S
Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia.
Krizaj
Igor
I
Rozman
Blaz
B
Kveder
Tanja
T
Bozic
Borut
B
D001323:Autoantibodies / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D018046:Protein C Inhibitor / Q000097:blood / Q000302:isolation & purification*; D053482:beta 2-Glycoprotein I
D001323:Autoantibodies; D006023:Glycoproteins; D018046:Protein C Inhibitor; D053482:beta 2-Glycoprotein I