Three novel high performance affinity chromatographic media for the separation of antithrombin III from human plasma

Abstract

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.

Methacrylate‐based chromatographic media

“…This stationary phase was then used for affinity chromatography of complementary ssDNA, which could hybridize with the immobilized sequence [125]. Similarly GMA beads with immobilized heparin were used for the separation of antithrombin III from human plasma [126]. are typically bound to the support via immobilized chelate forming ligand attached to the support and attract the separated compounds through their complexing units, typically histidine, tryptophan, cysteine, and phosphate groups.…”

A novel matrix for high performance affinity chromatography and its application in the purification of antithrombin III

“…The partially hydrolyzed heparin can be readily reacted with the epoxy groups of epichlorohydrin-viscose fibers. The maximum binding capacity to AT III on the heparin media made of this coupling method has been revealed in our previous work [16]. After coupling reaction, the excess of epoxy groups remained on the heparin-viscose fibers was deactivated by ethanolamine.…”

“…Heparin-immobilized media has been widely used as an affinity adsorbent for the separation and purification of plasma components in blood-coagulation systems [16,17]. Because viscose is lack of active group, the attachment of heparin is made in two steps: activation and coupling.…”

Specific DNA G‐quadruplexes bind to ethanolamines

“…The HPAC supports were prepared by immobilizing ammonia and ethanolamine on home-made monodisperse, nonporous, cross-linked polyglycidylmethacrylate (PGMA) beads (4 lm) bearing free epoxide groups with the density of 1.34 mmol g 21 beads. 31,32 The nonspecifically electrostatic interaction between protonated amino groups and DNAs (polyanion) may play a dominant role when using amino modified support to separate DNAs. To give prominence to the specific interaction of DNAs to modified ethanolamine, the affinity supports with low density of ethanolamines were prepared as shown in Figure 1.…”