Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma.
Novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were employed as the support for high performance affinity chromatography. Heparin was covalently attached to PGMA beads by three different coupling methods. Heparin-PGMA-I was prepared by directly coupling amino-groups of heparin with PGMA. Heparin-PGMA-II and III were prepared by the coupling of heparin to amino-PGMA, which was obtained by amination of PGMA. Heparin-PGMA-II was prepared by coupling the carboxyl groups of heparin to amino-PGMA by using water-soluble carbodiimide as coupling reagent, and heparin-PGMA-III was prepared by the reductive amination of heparin and amino-PGMA with sodium cyanoborohydride. The heparin contents of heparin-PGMA-I, II and III were 1.6, 10.2 and 1.0 mg/g beads, respectively. Their affinity capacities for antithrombin III were investigated. Their binding activity to antithrombin III was not proportional to the content of heparin immobilized, and heparin-PGMA-I was the most efficient affinity medium for antithrombin III. The resultant affinity media presented minimal non-specific interaction with other proteins and can be used in a wide pH range. All the three heparin-PGMA beads were exploited for the separation of antithrombin III from human plasma. The purity of antithrombin III obtained was higher than 90%, which was confirmed by high performance size exclusion chromatography.
Zhao
Y
Y
Center for Molecular Science, Institute of Chemistry, Chinese Academy of Science, Beijing 100080, People's Republic of China.
Zhao
R
R
Shangguan
D
D
Xiong
S
S
Liu
G
G
D000990:Antithrombin III / Q000032:analysis / Q000302:isolation & purification*; D001769:Blood; D002846:Chromatography, Affinity / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry; D006493:Heparin; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D008956:Models, Chemical; D011108:Polymers
D004852:Epoxy Compounds; D008689:Methacrylates; D011108:Polymers; D000990:Antithrombin III; D006493:Heparin; C007870:glycidyl methacrylate