Jan B. L. Damm

A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin

For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz1H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAcα2-3Galβ1-4GlcNAcβ1-2Manα1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-2Manα1-3]Man β1-4GlcNAcβ1-4[Fucα1-6]0-1GlcNAc and Manα1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-2Man α1-3]Manβ1-4GlcNAcβ1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAcα2-3Galβ1-3[NeuAcα2-6]GalNAc, NeuAcα2-3Galβ 1-3GalNAc and NeuAcα2-3Galβ1-3[NeuAcα2-3Galβ1-4GlcNAcβ1-6]GalNAc.

Separation of natural and synthetic heparin fragments by high-performance capillary electrophoresis.

The application of capillary electrophoresis (CE) for the analysis of natural and synthetic low-molecular-mass heparin fragments at low pH is described. It is demonstrated that under the applied conditions the separation is based on charge, charge distribution and molecular mass of the heparin molecules, yielding a high resolution. It is shown that the presence of sodium chloride in the sample solution has hardly any effect on the CE performance. However, the pH of the electrophoresis buffer is a critical parameter. The resolutions obtained with CE and high-performance anion-exchange chromatography (HPAEC) are compared for various heparin fragments and it is concluded that, at least for this type of molecule, CE forms an attractive alternative to HPAEC.

Structure of three acidic O-linked carbohydrate chains of porcine zona pellucida glycoproteins

Structural analysis by ID and 2D 1H NMR spectroscopy of three acidic O‐linked oligosaccharide alditols, released from porcine zona pellucida glycoproteins by alkaline borohydride treatment, afforded the following structures: Galβl‐4(6SO4‐)GlcNAcβl‐3Galβl‐4GlcNAcβ1‐3Galβ1‐3GalNAc‐ol Neu5Gcα2‐3Gal/91 ‐4(6SO4‐)GlcNAqβl ‐3Ga1βl ‐4GlcNAcβl ‐3Galβl ‐3GalNAc‐ol Neu5Acα2‐3Galβl ‐4(6SO4‐)GlcNAcβl ‐3Galβl ‐4GlcNAcβl ‐3Galβl‐3GalNAc‐ol These oligosaccharides are the smallest compounds that contain the structural elements which are present in the acidic, high‐molecular mass O‐linked carbohydrate chains of porcine zona pellucida glycoproteins.

Indirect UV detection as a non-selective detection method in the qualitative and quantitative analysis of heparin fragments by high-performance capillary electrophoresis

The application of capillary electrophoresis (CE) in combination with indirect UV detection for the qualitative and quantitative analysis of synthetic low-molecular-mass heparin fragments, at low pH, is described. It is demonstrated that, in contrast to direct UV detection, with indirect UV detection the signal obtained for various synthetic heparin pentasaccharides is nearly independent of their molecular structure. Moreover, the sensitivity of indirect UV detection is at least one order of magnitude higher than that of direct UV detection. CE-indirect UV detection for the qualitative and quantitative analysis of low-molecular-mass glycosaminoglycans was achieved by using 5 mM 5-sulphosalisylic acid, pH 3 or 5 mM 1,2,4-tricarboxybenzoic acid, pH 3.5 as electrophoresis buffer and chromophore. The technique is exemplified by the analysis of three pharmaceutical preparations of synthetic heparin pentasaccharides. The method employing indirect UV detection was validated with respect to repeatability, limit of detection, limit of quantitation, linearity, accuracy and ruggedness. In the indirect detection mode, the limit of detection for synthetic pentasaccharides is below 5 fmol, whereas the limit of quantitation is about 25 fmol. The method shows excellent repeatability and is linear in the femtomole-picomole range. Finally, it is demonstrated that the method is suitable for the analysis of various types of glycosaminoglycans.

Separation of sialyl-oligosaccharides by medium pressure anion-exchange chromatography on Mono Q®

On columns prepacked with the recently introduced anion-exchange material, Mono Q (Pharmacia), sialyl-oligosaccharides could be fractionated excellently according to the sialic acid content. With u.v. absorption at 214 nm as the detection method, analytical runs of carbohydrate material on the microgram scale, were possible. The value of the method for preparative purposes was demonstrated for sialic acid-containing carbohydrates obtained from human serotransferrin by hydrazinolysis.

The β-subunit of human chorionic gonadotropin contains N-glycosidic trisialo tri- and tri'-antennary carbohydrate chains

TheN-linked carbohydrate chains of theβ-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri′-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz1H-NMR spectroscopy to be