Concomitant isolation of protein C inhibitor and unnicked β2-glycoprotein I
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Saša Čučnik
Abstract
β2-Glycoprotein I (β2GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of β2GPI 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 β2GPI. In set No. 2 only unnicked β2GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked β2GPI in set No. 3. Oxidation or degradation of β2GPI during the isolation procedure did not result in a mixture of different forms of β2GPI but rather in a lower yield of wild-type β2GPI. The co-existence of β2GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.
Copyright © 2004 by Walter de Gruyter GmbH & Co. KG
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- Matrix metalloproteinases, inflammation and atherosclerosis: therapeutic perspectives
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