LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOLUME 1. THE FOUNDATIONS OF BIOCHEMISTRY, STRUCTURE AND CATALYSIS - 2011

PART I. STRUCTURE AND CATALYSIS

7. CARBOHYDRATES AND GLYCOBIOLOGY

7.5. Carbohydrate Analysis

The growing appreciation of the vital roles that Oligosaccharides play in biological recognition processes has spurred The Development of Methods for analyzing the Structure and stereochemistry of complex oligosaccharides. Oligosaccharide analysis is complicated by the fact that, unlike Peptides and Nucleic Acids, they can branch and join via multiple types of linkages. The high charge density of many Polysaccharides and oligosaccharides, combined with the relative lability of sulfate esters in glycosaminoglycans, only compounds these difficulties.

For simple linear polymers such as amylose, the positions of glycosidic bonds are determined by treating the starting polysaccharide with methyl iodide in a strongly alkaline environment. This methylates all free hydroxyl groups, rendering them acid-resistant, after which the methylated polysaccharide is acid-hydrolyzed. The free hydroxyl groups in the resulting Monosaccharide Derivatives are those that participated in the glycosidic bonds in the original polymer. To determine The sequence of monosaccharide units and the presence of branch points, exoglycosidases of known Specificity are employed: they sequentially cleave monosaccharide residues from the nonreducing end of the chain. The specificity of these exoglycosidases often makes it possible to determine the position and stereochemistry of each linkage.

To analyze The structure of the oligosaccharide portion of Glycoproteins and Glycolipids, the oligosaccharide moiety is cleaved using purified Enzymes. Glycosidases specifically cleave O- or N-oligosaccharide linkages, while lipases remove lipid "heads." In addition, O-linked glycans can be released from glycoproteins by Hydrazinolysis. The resulting sugar mixture can be separated into individual components using various methods (Fig. 7-36), including those used for PROTEIN AND AMINO acid Separation: fractional solvent precipitation, and ion-exchange and size-exclusion chromatography (see Fig. 3-17). Purified Lectins attached to a solid support are frequently used in Affinity Chromatography to fractionate sugar mixtures (see Fig. 3-17c).

Acid Hydrolysis of oligosaccharides and polysaccharides under strong acid conditions yields a mixture of Monosaccharides that can be identified using chromatographic methods, thereby establishing the overall COMPOSITION OF THE polymer.

Class="center">Fig. 7-36. Methods for carbohydrate analysis. Complete characterization of a carbohydrate purified in the initial stage sometimes requires the application of all four analytical approaches.

Oligosaccharide analysis increasingly relies on mass spectrometry and high-resolution NMR spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry can be used to analyze such polar compounds as oligosaccharides (Box 3-2). MALDI is sensitive enough to determine the mass of ions—in this case, entire oligosaccharide chains (Fig. 7-37). Tandem mass spectrometry determines the mass of both ions and their fragments generated by the Cleavage of glycosidic bonds. NMR data (Box 4-5) help determine monosaccharide sequences, linkage positions, and anomeric carbon configurations, especially for medium-sized oligosaccharides. For example, the STRUCTURE OF THE heparin segment whose spatial model is shown in Fig. 7-22 was entirely elucidated using NMR spectroscopy. Automated methods and specialized instruments are employed for routine oligosaccharide structure determination, yet sequencing a branched oligosaccharide with multiple types of linkages remains a far more challenging task than determining the linear sequence of a protein or nucleic acid.

instruments, but determining the sequence of a branched oligosaccharide with multiple linkage types remains a much more formidable challenge than determining the linear sequence of a protein or nucleic acid.

Fig. 7-37. Separation and quantitative analysis of oligosaccharides from a glycoprotein mixture. In this experiment, a mixture of Proteins isolated from Kidney tissue was treated to release oligosaccharide chains, which were then analyzed by mass spectrometry (MALDI-MS). Each oligosaccharide corresponds to a peak with a specific molecular mass, and the peak area reflects the Abundance of that oligosaccharide in the sample. The largest peak corresponds to an oligosaccharide with a mass of 2,837.4, consisting of 14 residues. Oligosaccharides containing 7 and 19 residues were also detected in the same sample.

Chemical synthesis plays a vital role in carbohydrate analysis, being widely used to study the BIOLOGICAL Functions OF glycosaminoglycans and oligosaccharides. Although the underlying chemistry is complex, modern methods allow the synthesis of short fragments of virtually any glycosaminoglycan with a desired stereochemical structure, length, and sulfate group arrangement, yielding oligosaccharides far more complex than those depicted in Fig. 7-29. Solid-phase oligosaccharide synthesis is based on the same principles as Peptide Synthesis and offers the same advantages (see Fig. 3-29). However, Carbohydrate Chemistry requires specialized techniques: protecting and activating groups must ensure The formation of glycosidic bonds with the correct orientation of hydroxyl groups. Purified glycosyltransferase enzymes are extremely useful for synthesizing chemically pure compounds. Such synthetic methods are currently of great interest because isolating and purifying specific oligosaccharides from natural sources is exceptionally difficult. Oligosaccharide microarrays and fluorescently labeled lectins can be used to determine binding specificity (analogous to the DNA Microarrays described in Chapter 9).

Summary of Section 7.5 Carbohydrate Analysis

■ Complete elucidation of an oligosaccharide or polysaccharide structure requires determining branch points, the monosaccharide sequence in each branch, the configuration of each monosaccharide unit, and the positions of glycosidic bonds—a task vastly more complex than protein or nucleic acid sequencing.

analysis of a protein or nucleic acid sequence.

■ The structure of oligosaccharides and polysaccharides is typically determined by combining several methods: specific Enzymatic hydrolysis helps establish the Stereochemistry of the compound and yields smaller fragments for analysis; methylation and acid hydrolysis allow the localization of glycosidic bonds; sequential degradation provides information on the monosaccharide sequence and the configuration of anomeric carbon atoms.

■ Mass spectrometry and high-resolution NMR spectrometry extract a wealth of information from small sample amounts regarding chain sequence, the configuration of anomeric and other carbon atoms, and glycosidic bond positions.

■ Solid-phase synthesis methods make it possible to produce oligosaccharides with predetermined structures, which is crucial for investigating oligosaccharide-lectin interactions and may also hold significant clinical importance.