BC367L_5_2011

BC367L_5_2011 - Experiment 8 DETERMINATION OF...

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Oligosaccharide Structures Page 1 of 5 Experiment 8 DETERMINATION OF OLIGOSACCHARIDE STRUCTURES Introduction The structures of carbohydrates have many important consequences in biochemistry. In order to understand the complex nature of oligosaccharide linkages this lab will introduce you to the structural determination of an unknown carbohydrate, and allow us to determine some enzyme specificity as well. The following oligosaccharides will be available in this laboratory. One of these carbohydrates will be your unknown sample, and the others will be used as controls for different chemical and enzymatic tests. Name Structure Specific Optical Rotation [ ] D (° mL dm -1 g -1 ) Cellobiose Glc( β 1 4)Glc 35.0 Lactose Gal( β 1 4)Glc 52.0 Maltose Glc( α 1 4)Glc 130.4 Melezitose Glc( α 1 β 2)Fru(3 ± α 1)Glc 88.0 Melibiose Gal( α 1 6)Glc 129.5 Raffinose Gal( α 1 6 29 Glc( α 1 β 2)Fru 105.2 Sucrose Glc( α 1 β 2)Fru 66.5 Trehalose Glc( α 1 α 1 29 Glc 178.0 Turanose Glc( α 1 3)Fru 75.8 CHEMICAL TESTS FOR REDUCING SUGARS Carbohydrates can be classified as either reducing or nonreducing sugars. A reducing sugar will have a free aldehyde group or hemiacetal group, but keto or hemiketal groups are also classified as reducing sugars because under alkaline conditions, they can isomerize to aldehydes or hemiacetals. If an oxidizing agent is added to an alkaline solution with a reducing sugar, the hemiacetal is oxidized to the corresponding acid, and the oxidizing agent is subsequently reduced. A common oxidizing agent for carbohydrates is an alkaline solution of copper(II) (Cu 2+ ) known as Benedict’s reagent. Reduction of Cu 2+ to Cu + is readily visualized by the precipitation of a brick-red cuprous oxide (CuO 2 ) in alkaline solution. Hemiacetal + Cu 2+ Acid + CuO 2 alkaline ACID HYDROLYSIS OF OLIGOSACCHARIDES Acetal and ketal bonds that link oligosaccharides are hydrolyzed in acidic solution to produce the monosaccharide components. One would expect that acid hydrolysis would be a nonspecific method of disrupting oliogsaccharide linkages. However, the kinetics of hydrolysis in oligosaccharides is bond specific and a partial acid hydrolysis experiment can thus reveal more
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Oligosaccharide Structures Page 2 of 5 details than simply the carbohydrate composition. For example, the α - β -acetal-ketal linkage found in sucrose is the most readily hydrolyzed and the α - α -acetal-acetal found in trehalose is the most stable of the oligosaccharide linkages. Sucrose + H + Glucose + Fructose ENZYME HYDROLYSIS The acetal and ketal linkages of oligosaccharides can also be cleaved by enzymatic hydrolysis. Enzymes can vary widely in their specificity for a particular linkage, and in this way they can be used to characterize the α or β nature of bonding within an oligosaccharide. Three different enzymes will be used in this experiment (
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BC367L_5_2011 - Experiment 8 DETERMINATION OF...

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