Characterization of antihrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

Leopold Kremser, Andrea Brükner, Andrea Heger, Thomas Grunert, Andrea Buchacher, Djuro Josic, Günter M Allmaier, Andreas Rizzi

Veröffentlichungen: Beitrag in FachzeitschriftArtikelPeer Reviewed

Abstract

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and p/ differences down to 0.01 pH units could be distinguished. ATIII-a and ATIII-ß-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-a and ATIII-ß exhibit p/ values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The p/ difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-ß. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-a main isoform and about 6.6% of ATIII-ß. The p/ values of ATIII determined by CIEF with internal calibration were in fair agreement with the p/ values of the main isoforms achieved with 2-DE.
OriginalspracheEnglisch
Seiten (von - bis)4282-4290
Seitenumfang9
FachzeitschriftElectrophoresis
Jahrgang24
Ausgabenummer24
DOIs
PublikationsstatusVeröffentlicht - 2003

ÖFOS 2012

  • 1040 Chemie

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