chem

• Title of exer 8 - Glycogen ISOLATION and purity determination
• Glycogen is the reserve polysaccharide of animals. It consists of D-glucose units linked together by a-1,4- and a-1,6-glycosidic linkages.
• Title of exer 8 - Glycogen ISOLATION and purity determination
• Glycogen has been isolated from liver, skeletal and cardiac muscles of mammals, and has been detected in cells of all animals. The liver of a well-fed human may contain glycogen up to 10% by mass. This polysaccharide is also present in tissues of invertebrates, bacteria and protozoa
• Early extractions of glycogen involve heating the sample with 30% aqueous solution of potassium hydroxide at 100°C for three hours. Glycogen is isolated by solvent precipitation with ethanol, Purification is done by repeated precipitation with ethanol from aqueous solution. Degradation of glycogen occurs under this harsh condition; hence, newer methods have been developed.
• Attempts to isolate glycogen in its native state have led to formulation of several extraction methods. Glycogen can be extracted from a sample with cold, dilute trichloroacetic acid (TCA). The molecular weight of glycogen from this method is ten times than that of glycogen from previous preparations. However, the molecular weight range of the glycogen isolated by this method is broad, owing to partial degradation
• Another method uses dimethylsulfoxide (DMSO) as an extracting solvent. This gives a glycogen preparation with higher molecular weight and less polydispersity, although the yield is lower. This may show that DMSO preferentially extracts the easily accessible glycogen with narrow molecular weight range and leaves behind the metabolically active, low molecular weight, residual glycogen.
• Cold water, aqueous phenol, glycine buffer and aqueous mercuric chloride can also be used as extracting solvents. Evidences suggest that these newer extraction procedures yield glycogen samples that are representative of the polysaccharide in its native state. If native glycogen is desired, hot solvents, acids, and alkalis should be avoided
• Isolation of carbohydrates from tissue samples involves extraction with aqueous solvent and removal of proteins and nucleic acids. A commonly used reagent in the removal of proteins is trichloroacetic acid (TCA). Although large proteins and other polysaccharides are not extracted by aqueous TCA, there are a number of other impurities included in the carbohydrate isolate. Amino acids, oligosaccharides and some salts may be included in the sample. The purity of the product is necessary to gauge the actual amount of carbohydrate present.
• To determine the purity of the glycogen isolate, the sample will be hydrolyzed by acid into its monomer units and the amount of glucose formed from the sample after hydrolysis can be determined quantitatively using the Nelson's method for reducing sugars.
• The actual umodse of glucose in the glycogen sample can be determined from the amount of glucose released (in umol) upon acid hydrolysis of the glycogen sample. This is determined using the Nelson's method.
• Actual µmol glucose = µmol glucose in the hydrolyzed sample
• Theoretical µmol of glucose in the glycogen sample


  Can be calculated using a conversion factor
• Conversion factor

  Based on the molar mass of a free glucose unit and a glucose residue in glycogen
• Molar mass of free glucose

  180 g/mol
• Formation of a glycosidic bond between glucose units


  Involves the removal of 1 mol of water for every mol of glucose residue in the polysaccharide chain
• Molar mass of a glucose residue in glycogen

  162 g/mol (180-18)
• The theoretical µmol of glucose in the glycogen sample can be calculated using a conversion factor, which is based on the molar mass of a free glucose unit and a glucose residue in glycogen. The molar mass of free glucose is 180 g/mol. In a glycogen molecule, glucose units are joined together by glycosidic bonds. Formation of a glycosidic bond between glucose units involves the removal of 1 mol of water for every mol of glucose residue in the polysaccharide chain
• The mass of glycogen in equation 8-3 is the amount of glycogen used in the experiment, which is also equal to the mass of glucose residues present in the glycogen sample. The factor 180/162 will convert the mass of a glucose residue into the mass of a free glucose. It is the amount of free glucose that is being determined in the experiment using the Nelson's method. The factor 1/180 will convert the amount of free glucose from mg to mmol. The factor 1000 is used to convert mmol free glucose to umols
• Like glycogen, starch is a polymer consisting entirely of D-glucose units. Two starch fractions, amylose (10-20%) and amylopectin (80-90%), can usually be isolated from plants. Structurally, glycogen is very similar to amylopectin, which both contains (1-4) and a(1-6) linkages between glucose units. Thus, equation 8-1 can also be used to determine the purity of an amylopectin sample
• 30% aqueous KOH


  Strong base that can solubilize tissue and saponify fat, contributing to extracting pure glycogen upon application of ethanol facilitating solvent precipitation
• Cold aqueous trichloroacetic acid (TCA)


  Eliminates proteins and nucleic acid by promoting the precipitation of macromolecules, but does not promote precipitation to smaller molecules such as glycogen, thus promoting the isolation of glycogen from a solution with other macromolecules
• Ethanol


  Precipitates glycogen by dehydrating it, as ethanol can interact more with water causing the interaction of water and glycogen to weaken allowing precipitates of glycogen to occur
• Factors affecting glycogen yield


  • Purity of isolated glycogen (removal of contaminants like proteins and lipids)
  • Extraction method and technique (enzymatic digestion or chemical extraction)
  • Characterization method (enzymatic assays or colorimetric techniques)
• The purity of the isolated glycogen, which refers to the removal of contaminants such as proteins and lipids, can affect the accurate quantification of glycogen
• The extraction method and technique used, such as enzymatic digestion or chemical extraction, can influence the yield by affecting the efficiency of glycogen recovery from tissues
• The characterization method employed to quantify glycogen, whether through enzymatic assays or colorimetric techniques, plays a crucial role in determining the measured yield, as different methods may have varying sensitivities and accuracies in detecting glycogen levels