Glycoproteins immobilization

Bovine serum albumin covalently bonded to silica and a-acid glycoprotein immobilized on silica have been used to resolve a wide range of acidic and basic drugs and amino acid derivatives [807-810]. Because of their complex structures, however, the... 

Lectin affinity chromatography may be used to purify glycoproteins Immobilized antibodies may be used as affinity absorbants for the antigen that stimulated their production (e.g. purification of factor VIII using immobilized anti-factor VIII antibodies)... 

Karlsson and Hermansson [30] used chemometrics for optimization of chiral separation of omeprazole and one of its metabolites on immobilized al-acid glycoprotein. Plasma was centrifuged at 2500 rpm and a portion (20-50 ji ) was injected into a 5-/rm Chiral-AGP column (10 cm x 4 mm) with al-acid glycoprotein immobilized to silica as a chiral stationary phase and acetonitrile-phosphate buffer of pH 5.7-7.2 as mobile phase (1 ml/min). Detection of omeprazole and its main metabolite, hydroxy-lated omeprazole, was performed at 302 nm. A statistical model was developed for the optimization of the operational parameters. The experimental data were evaluated with multivariate analyses column temperature and acetonitrile concentration were the most important variables for the enantioseparations. Complete enantiomeric separation for omeprazole and hydoxylated omeprazole was obtained within 15 min. 

Chiral-AGP (10 cm X 4 mm, 5 jim) packed with a 1-acid glycoprotein immobilized on silica... 

EY Laboratories, San Mateo, USA Carbohydrates, glycoproteins (immobilized lectins)... 

Proteins. A chiral stationary phase with immobilized a -acid glycoprotein on silica beads was introduced by Hermansson in 1983 [18, 19]. Several other proteins such as chicken egg albumin (ovalbumin), human serum albumin, and cellohy-drolase were also used later for the preparation of commercial CSPs. Their selectivity is believed to occur as a result of excess of dispersive forces acting on the more retained enantiomer [17]. These separation media often exhibit only modest loading capacity. 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

D.J.O. Shannessy and W.L. Hoffman, Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotechnol. Appl. Biochem. 9, 488—496 (1987). 

Donohue-Rolfe, Arthur, David W.K. Acheson, Anne V. Kane, and Gerald T. Keusch. "Purification of Shiga Toxin and Shiga-Like Toxins I and II by Receptor Analog Affinity Chromatography with Immobilized PI Glycoprotein and Production of Cross-Reactive Monoclonal Antibodies." Infection and Immunity 57 (December 1989) 3888-893. 

Table 6.4 Some lectins commonly used in immobilized format for the purification of glycoproteins. The sugar specificity is listed, as are the free sugars used to elute the bound glycoprotein...

Fig. 10.15 Virus detection test. Sensor signal (phase change) measured between channel 1 and the reference channel for the immobilization of anti HSV 1 glycoprotein G monoclonal antibody layer on the sensing surface of channel 1 (A HSV i gG) and the binding of HSV 1 particles to this layer (A IISV i). Reprinted from Ref. 28 with permission. 2008 American Chemical Society...

Williams and Wainer (2002) use examples of two chiral separations to demonstrate their utility in research. In one example, the difference between enantiomers in the competitive displacement of cyclosporine from immobilized P-glycoprotein was studied. In the other, the pharmacokinetic profiles of (+) and (—)-ketamine and (-h) and (—)-norketamine were determined (Williams and Wainer, 2002). 

Y Zhang, F Leonessa, R Clarke, IW Wainer. Development of an immobilized P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities. J Chromatogr B 739 33-37, 2000. 

For the modification of the gold surface different chemical and biological substances (dodecanethiol, lectins, dextran sulfate DS, with the mass of 5 kDa and polyelectrolytes) were used. The mercaptane layer was formed in ethanol atlO" M concentration of thiol. Polyaniline hydrochloride (PAH), Aldrich , USA, was used to cover the transducer surface by a water insoluble polymer. Lectins from Phaseolus vulgaris (PLA), Solanum tuberosum (STA), Helix pomatia (HPA) and Tuberosum vulgaris (WGA) were immobilized on the surface pre-treated with dodecanethiol or PAH. These lectins were used for the immobilization of glycoproteins. Blocking of the free binding sites was carried out with 1% bovine serum albumin (BSA). 

Lectins have been used for purifying glycoproteins present in cell membranes. The lectin of wheat germ was immobilized on agarose ac-... 

Comparable to the other protein-coated supports are the Ultrabiosep and the BioTrap phases. The former are composed of C4, Cg, or Cis reversed-phase silica supports covered with a biological polymer which is not described in the literature (135). The latter are commercially available as Bio Trap Acid or Biotrap Amine precolumns (136). They are Cis-modified silica supports covered with -1-acid glycoprotein as a biocompatible layer. Due to the immobilized protein, this type of reversed-phase material also possesses weak ion-exchange properties. 

---