Isolation of Glycogen and Purity Determination J. Bernido, J.

Vicente University of the Philippines Visayas Miagao,Iloilo, Philippines

ABSTRACT: This experiment generally aims to study the polysaccharide Glycogen. Specifically, this aims to isolate glycogen from mussel flesh and chicken liver by virtue of its difference in terms of physical and chemical properties from other biomolecules. Also this aims to purify the isolated glycogen from the two different samples. The purity of the isolated glycogen was determined using colorimetric analysis after acid hydrolysis and treatment with Nelsons Reagent. Results revealed that we have successfully extracted glycogen from the sample tissues. Among all the test conditions done, the one with the highest exposure time to heating had the highest concentration of glucose.

1. Introduction The brain and other tissues require a constant supply of blood glucose for survival. Glucose from the diet, though, arrives irregularly. Some tissues, particularly the liver and skeletal muscle, store glucose in a form that can be rapidly mobilized, glycogen. Liver glycogen is used to buffer the overall blood glucose level; glycogen is synthesized when blood glucose is high, and glycogen is degraded (with the resulting glucose released into the blood stream) when blood glucose is low, such as during the early stages of a fast. Muscle uses its glycogen stores for energy during strenuous exercise. Glycogen is a molecule that serves as the secondary longterm energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue. Glycogen is made primarily by the liver and the muscles, but can also be made by glycogenesis within the brain and stomach.

As seen in Figure 1, Glycogen has a complex molecular structure. It has a core protein of glycogenin is surrounded by branches of glucose units. The entire globular granule may contain approximately 30,000 glucose units. Glycogen is the analogue of starch, a less branched glucose polymer in plants, and is sometimes referred to as animal starch, having a similar structure to amylopectin. Glycogen is found in the form of granules in the cytosol/cytoplasm in many cell types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose, but one that is less compact than the energy reserves of triglycerides (lipids).

2.Theory
Isolation of Glycogen The physical and chemical properties of many neutral polysaccharides are sufficiently different from those of other naturally occurring substances to permit their ready isolation. Thus, when clam or mussel flesh is homogenized by saponification and subsequently treated with trichloroacetic acid. Many high molecular weight compounds, such as proteins and nucleic acids, are readily precipitated while glycogen remains in the solution. Since polysaccharides are considerably less soluble than sugars and aqueous alcohol, glycogen can be separated from sugars and other watersoluble compounds by precipitation with alcohol. Purified glycogen is obtained from aqueous solution by subsequent reprecipitation with alcohol.

Figure 1. Schematic Cross-sectional view of glycogen

Structure of glycogen Glycogen is a chain of glucose subunits held together by 1,4--glycosidic bonds, very much like starch (a-amylose). In contrast to starch, which is a single linear chain of glucose, glycogen is a branched structure. At the branch points, subunits are joined 1,4--glycosidic bonds. Branches occur every 8-10 residues of glucose. Glycogen synthesis Glycogen synthesis is, unlike its breakdown, endergonic. This means that glycogen synthesis requires the input of energy. Energy for glycogen synthesis comes from UTP, which reacts with glucose-1-phosphate, forming UDP-glucose, in reaction catalysed by UDP-glucose pyrophosphorylase. Glycogen is synthesized from monomers of UDP-glucose by the enzyme glycogen synthase, which progressively lengthens the glycogen chain with (14) bonded glucose. As glycogen synthase can lengthen only an existing chain, the protein glycogenin is needed to initiate the synthesis of glycogen. The glycogen-branching enzyme, amylo (14) to (16) transglycosylase, catalyzes the transfer of a terminal fragment of 6-7 glucose residues from a nonreducing end to the C-6 hydroxyl group of a glucose residue deeper into the interior of the glycogen molecule. The branching enzyme can act upon only a branch having at least 11 residues, and the enzyme may transfer to the same glucose chain or adjacent glucose chains. Glycogen Metabolism Glycogen is cleaved from the nonreducing ends of the chain by the enzyme glycogen phosphorylase to produce monomers of glucose-1-phosphate, which is then converted to glucose 6phosphate by phosphoglucomutase. A special debranching enzyme is needed to remove the alpha(1-6) branches in branched glycogen and reshape the chain into linear polymer. The G6P monomers produced have three possible fates:

Hydrolysis and Purity Determination of Glycogen

Figure 3. Hydrolysis of Glycogen into Glucose Molecules Like other polysaccharides (starch, and cellulose), glycogen can also undergo hydrolysis. During the reaction, the glucose monomer units of Glycogen are separated. This is being done by the introduction of water in the glycogen molecule with the presence of strong acid or a base. The reaction is summarized in Figure 3. In this experiment, glycogen was hydrolyzed by strong acid to give an estimate of the polysaccharide content of the sample. This was done by determining the glucose content of the glycogen after the hydrolysis. The Nelson Test was used in the quantitative determination of the glucose since it is both sensitive and reproducible. In this analysis, the sugar was heated with an alkaline copper reagent resulting in the formation of the rust colored precipitate, cuprous oxide. The amount of cuprous oxide produced was then determined colorimetrically by the addition of arsenomolybdic acid, which is quantitatively reduced to arsenomolybdous acid by cuprous ion. The intense blue color of the arsenomolybdous acid is determined colorimetrically as a quantitative measure of the glucose content.

3.Methodology
A. Isolation of Glycogen Figure 2. Cleaving action of Glycogen Phosphorylase on the Glycogen. With the enzymatic action of Glycogen Phosphorylase, Huge Glycogen Molecules are broken down into its smaller monomer units of glucose. The glucose molecules can now then be used by the cell for energy production. The mussel flesh was first washed thoroughly and homogenized using osterizer. Twelve mL of boiling water was poured to each 3 g of osterized mussel flesh. The mixture was stirred and then boiled for 2 min. Three mL of distilled water was added to the mixture which was subsequently heated in a boiling water bath for 30 min. The mixture was then filtered after being added with 1 mL of 0.1% CH 3COOH. The filtrate

was then immersed in an ice bath for 15 minute to allow precipitation. The solution was then centrifuged and the precipitate was collected. B. Standard Curve for Glucose A 0.5 mM glucose solution was prepared analytically. Aliquots of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL were placed separately in a test tube calibrated to 10 mL. Calibration was done by pipetting out 10.0 mL of water into the test tube and marking the lower meniscus. A blank of 1.0 mL distilled water was prepared and 1.0 mL of Nelsons reagent was added to all 7 test tubes. Nelsons reagent should be prepared freshly before use by combining 50 mL of reagent A and 2 mL reagent B. A marble was placed on top of each tube and heat in a boiling water bath for 20 mins. The tubes were cooled in a beaker of tap water. When cooled, 1.0 mL of arsenomolybdate reagent was added. The tubes were mixed and allowed to stand for a few minutes. Distilled water was added to 10 mL mark, mixed well, and read the absorbance at 510 nm. C. Acid Hydrolysis Fifty milligrams of the crude glycogen isolate was dissolved in 5 mL distilled water and 0.4 ml aliquot of the solution was added to each of seven numbered test tubes (test tubes numbers 2 to 8). Tube no. 1 was filled with 0.4 mL distilled water and used as a blank. A volume of 0.6 mL of 2N HCl was added to each tube and mixed well. One mL of 1.2N NaOH was added to tubes 1 and 2 and set aside at room temperature. Tubes 3 to 8 were placed in a boiling water bath. Tubes 3 to 7 were removed from the water bath at 4-minute intervals and the content was neutralized each time by adding 1 mL 1.2N NaOH. Tube 8 was allowed to boil for 30 mins and neutralized with the same amount of base. Each tube was diluted to the 10 mL mark. From this mixture, 0.5 mL aliquot was taken and diluted to 1.0 mL. Each solution was mixed well and assayed including blank using Nelsons method.

We have successfully extracted from the Mussel flesh. It was a white powdery substance as shown in Figure 4. Though there were some revisions in the methods the extracted glycogen from the sample was still isolated from impurities, especially proteins, since we still included the protein precipitation. The precipitation of the proteins was done by boiling the solution. During heating, glycogen was left soluble in the solution while proteins were denatured and precipitated. The precipitation process was enhanced by the addition of 0.1% CH3COOH, The impurities or precipitate was separated from the solution by filtration.

Figure 5. Hydrolysis and Nelsons Method Treatment

After isolation, the glycogen was then analyzed for its purity using colorimetric analysis. This was done by hydrolyzing the glycogen with strong acid. After which the resulting products were treated using the Nelsons method. The resulting solution was deep-blue colored (see Figure 5).

4. Results and Discussions

Test Solutions 1 2 3 4 5 6 7 8

Abs 0.82 0.83 0.88 0.97 0.99 1 1.09 1.1

Concentration (mM) ND ND ND 0.00883 0.0109 0.0119 0.0210 0.0221

Figure 4. Crude Glycogen

Table1. Summary of the results obtained for the determination of the purity of glycogen

From the standard calibration curve generated (see Figure 6), the concentrations of the sample test solutions under different conditions were calculated. Table 1 summarizes the results obtained for the determination of the purity of the isolated glycogen. Unfortunately, three of the sample solutions were not able to be calculated since they did not fall within the range of the standard calibration curve. These three solutions were the two solutions treated at room temperatures (Test tube 1 and 2) and the solution having the least period of exposure to boiling water (Test tube 3). Those which were detected, were the solutions treated at boiling water. Among all the detected solutions, the one which had the longest period of exposure to boiling water (Test tube8) had the highest concentration. It had a concentration of 0.0221 mM.

include Benedicts test, Barfoeds test, Seliwanoffs test, Bials Orcinol test, Mucic Acid test, Phenylhydrazone test. In a way, we would incorporate our knowledge about sugars with this experiment

Acknowledgement
We would like to give thanks to our course professor Dr. Vivian Topor for her time, effort, and patience in sharing us her knowledge which made the success of this experiment. We also want to acknowledge ourselves for enabling to finish this challenging, long, yet fun experiment. To the other groups for collaborating in some problems which made it easier for us to solve, thank you. Last but definitely not the least, we also give all our praises to our Heavenly Father for his omnipotent blessing to us.

Standard Calibration Curve

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

References
P. Champe, R. Harvey, D. Ferrer, Lippincolts Illustrated Reviews: Biochemistry. 4th Edition.

Absorbance

y = 9.8243x + 0.8833 R = 0.9958

0.02

0.04

0.06

Concentration (mM)

Figure 6. Standard Calibration Curve for the purity determination of Glycogen

5. Conclusions
From the results, data, and observations we have gathered, we were able to conclude that we have successfully isolated glycogen from the flesh of mussel. We have confirmed this using the determination of its purity using the colorimetric analysis of its hydrolysis product which is glucose via Nelsons method. Lastly, we would say that the amount of pure glycogen in our crude glycogen has a very low concentration with a maximum at only 0.0221 mM. We would like to recommend more confirmatory test for the presence of glycogen. This could include the general qualitative test like Molischs Test. It would also be better if we include specific test for any presence of Fructose, Lactose, Glucose, Xylose, Galactose, and Sucrose, etc. These tests