BCMB8020 April 11 2006 Requirements for Structural Determination of a Carbohydrate O O A O B C Identification of sugars Stereochemistry of each sugar Types of linkages Types of ring structures Anomeric configuration of each sugar Sequence of the different sugar residues 1 Methods to Determine Carbohydrate Structure The structural characterization of carbohydrates glycoconjugates is challenging due to the complexity diversity of carbohydrate structure To completely characterize an oligo or polysaccharide the following must be done determined 2 Methods to Determine Carbohydrate Structure 1 Isolate pure oligo polysaaccharide glycoconjugate 2 Cleave the carbohydrate s from the aglycone 3 Determine glycosyl residue composition 3 Methods to Determine Carbohydrate Structure 4 Determine absolute configuration i e D or L of each glycosyl residue specific rotation c d absorption enzymatic susceptibility derivatization and M S NMR 4 Methods to Determine Carbohydrate Structure 5 Determine glycosyl linkage s methylation analysis GCMS GLC MS 5 Methods to Determine Carbohydrate Structure 6 Establish ring form i e furanose or pyranose of each glycosyl residue methylation analysis ethylation 6 Methods to Determine Carbohydrate Structure 7 Determine sequence of the glycosyl residues multiple approaches 7 Methods to Determine Carbohydrate Structure 8 Determine the anomeric configuration i e or of each glycosyl residue NMR enzyme susceptibility 8 Methods to Determine Carbohydrate Structure 9 Determine points of attachment of any non carbohydrate substituents 10 Determine higher levels of structure when applicable 9 Purification of Carbohydrates The preparation of a purified carbohydrate or glycoconjugate may be challenging Carbohydrates can be separated from proteins lipids by aqueous organic extraction Aqueous Carbohydrates nucleic acids proteins Organic lipids Glycolipids glycoproteins are more difficult to purify Specific enzymes are often used to cleave the carbohydrate from the aglycone Oligo and polysaccharides can be further purified by standard chromatography i e ion exchange size exclusion HPLC preparative electrophoresis electrofocusing etc 10 Detection of Carbohydrates Colorimetric Assays for Carbohydrates Often done in microtitre plates compared to standard curves Sensitivity in the nanomole range General Carbohydrate Assay Phenol Sulfuric Acid Assay Sample 5 phenol H2SO4 Read OD at 490 nm Not good for amino and acetylated sugars Dubois M et al 1956 Anal Chem 28 350 356 Ferricyanide Assay Hydrolyze samples in 2N HCl for 2 hr at 100 C Treat with reducing agents Read OD at 690 nm Park Johnson 1949 J Biol Chem 181 149 11 Reducing sugar assays Para Hydroxybenzoic Acid Hydrazide PAHBAH Assay Add reagent boil 10 min Read OD 410 nm Lever 1972 Anal Biochem 47 248 Nelson Somogyi Assay Heat samples add reducing agent heat Read absorbance at 500 nm Nelson 1944 J Biol Chem 153 375 380 Somogyi 1952 J Biol Chem 195 19 23 Spiro 1966 Methods Enzymol 7 3 26 Green et al 1989 Anal Biochem 182 197 199 12 Hexose Assays Anthrone Assay Samples 0 2 anthrone in H2SO4 Boil Read OD at 620 nm Color may vary with type of sugar Dische A 1962 In Methods Carbohydr Chem 1 478 512 Uronic Acids Meta hydroxybiphenyl Assay for uronic acids Hydrolyze in boiling H2SO4 5 min add colorimetric reagent Read at 520 nm Blumenkrantz Asboe Hansen 1973 Anal Biochem 54 484 York et al 1985 Methods in Enzymology 118 3 40 see pg 26 Orcinol Assay for pentoses uronic acids Add colorimetric reagent HCL boil 20 min Read OD 665 nm Dische 1953 J Bio Chem 204 983 13 KDO Assay Thiobarbituric Acid TBA Assay for KDO and Sialic Acids Treat with periodate followed by colorimetric reagent Read OD 549 nm Aminoff 1961 Biochem J 81 384 Uchida 1977 J Biochem 82 1425 Sialic Acid Assays Resorcinol Assay for Sialic Acid Add colorimetric reagent in HCl boil 15 min add methylbutanol centrifuge Read upper phase at OD 450nm 580 nm Correct for hexose OD 450 Svennerholm 1959 Biochim Biophys Acta 24 604 611 Beeley 1985 In Laboaratroy techniuqes in biochm Mol Biol Vol 16 Elsevier 14 Other types of carbohydrate detection Electrochemical Detection DIONEX good sensitivity Refractive Index poor sensitivity If carbohydrate is derivatized UV or fluorescence Radioactive 15 Glycosyl Residue Composition Analysis Determination of the molar ratio of the constituent monosaccharides Basic steps 1 Cleave glycosidic bonds e g hydrolysis or methanolysis or solvolysis by hydrogen fluoride Note glycosidic bonds differ in their susceptibility to acid hydrolysis depends on monosaccharide and on position and anomeric configuration of glycosic linkage 16 2 Separate monosaccharides Four common methods are A Preparation of Alditol acetates GC can not distinguish uronic acids B Preparation of Trimethylsily TMS methylglycosides GC Useful to detect uronic and amino sugars Spectrum more complex than alditol acetate get anomer and maybe pyranose furanose C Separate by high pH anion exchange chromatography HPAEC D Monosaccharide composition analysis by Carbohydrate Electrophoresis FACETM FluorophoreAssisted Carbohydrate Electrophoresis 17 Preparation of alditol acetates GC composition analysis Glucose Hydrolyzed monosaccharide Glucitol Reduced monosaccharide NaBD4 NH4OH Remove NaBD4 borate with methanol acetic acid Add acetic anhydride base volatile alditol acetate Separate by GC NOTE can not specifically detect acidic sugars with this protocol 18 Preparation of Alditol Acetates OH O O OH H HO HO O HO OH OH OH NaBD 4 CHDOAc COAc AcOCH COAc CHOAc Ac2O Pyr OH OH HO HO CHOH OH D CH2OAc 19 GLC Profile of Alditol Acetates Glc 5 Hep Inos Gal Man Xyl Rib Ara Rha Fuc Detector Response Supelco SP2330 Column 10 Minutes 20 Preparation of Trimethylsilyl TMS Methyl Glycosides This methods does allow detection of acidic sugars Preparation of Trimethylsilyl TMS Methyl Glycosides O O O C OH OH O C OH H CH3OH HO OH HO O OCH3 O HO OCH3 HO C OCH3 OH O HO OH OH OCH3 CH3 3SiX O TMSO C O OCH3 OTMS O TMSO OCH3 OTMS TMSO C OCH3 OTMS O furanose forms TMSO OTMS OCH3 21 GalA Glc GlcA Hep Gal 10 Hep Glc Inos GlcA GalA Gal Xyl Man Fuc Ara Detector Response Rha GLC of TMS Methyl Glycosides J W Scientific DB1 column 20 Minutes 22 Separate by high pH anion exchange chromatography HPAEC Chromatography often done on a DIONEX HPLC system using a anion exchange column e g CarboPacPA1 with Pulsed amperometric PAD or electrochemical ED detection Since monosaccharides area weak acids pKa s 12 2 13 8 an alkaline eluent usually NaOH