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Experiment 6: Isolation of Glycogen and Glycogen Purity Determination

Introduction
Carbohydrates are one of the four major biomolecules along with proteins, lipids
and nucleic acids. As the most abundant organic molecule in nature, carbohydrates or
saccharides perform a range of functions which includes: providing a significant
fraction of dietary calories; supplying carbon for the synthesis of cell components;
serving as a form of stored chemical energy; and, forming part of the structures of
some cells and tissues (Ferrier, 2014; Seager and Slabaugh, 2011).
Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield
such compounds upon hydrolysis (Seager and Slabaugh, 2011). The empirical formula
for simple carbohydrates is (CH 2O) n , where n 3 (Ferrier, 2014). Carbohydrates are
classified to the following classes: Monosaccharides which contain only a single
polyhydroxy aldehyde or ketone unit; disaccharides which consists of two
monosaccharide units linked by a glycosidic bond; oligosaccharide which contains
three to ten monosaccharide units; and lastly, polysaccharides which contains very
long chains of hundreds or thousands of monosaccharide units (Seager and Slabaugh,
2011).
Polysaccharides are can either be storage or structural polysaccharides. Structural
polysaccharides give structure to plants and animals. Examples of structural
polysaccharides include cellulose in plants and chitin in the shells of crustaceans.
Structural polysaccharides are a storage form of energy. Examples of storage
polysaccharides include starch formed in roots and seeds as a form of glucose storage
and glycogen formed in the liver, also as a form of glucose storage.
In this experiment, glycogen found in the chicken liver is isolated and
characterized. Glycogen (Figure 1) is a branched glucan, that is, a branched polymer

consisting of linear segments of -D-glucopyranosyl units joined by -D-(14)


glycosidic linkages with -D-(16) glycosidic linkages joining the segments at the
branch points (BeMiller, 2011).
Figure 1. Glycogen (BeMiller, 2011)

Glycogen is an amorphous polymer, that is, it has no definite structure. This is


because it exists in a dynamic state. Considering its high molecular weight, glycogen
is highly soluble and exhibits an ideal hydrodynamic behavior. Just like all
polysaccharides, glycogen is also polydisperse especially since it is moving. Glycogen
stores D-glucose as an energy source until it is needed by the body. It also serves as a
buffering molecule to control the concentration of D-glucose in blood. These
properties result to difference of molecular weights of glycogen within a given tissue
(BeMiller, 2011).
In this experiment, glycogen is isolated based on the fact that these molecules are
resistant to the hydrolytic activity of an alkaline solution at increased temperature.
Interactions such as peptide bonds in proteins, esters bonds in lipids, and
phosphodiester bonds in ribonucleic acid are hydrolysed at these conditions, leaving
the glycogen intact separated from its impurities. Glycogen is then retrieved by
precipitation using ethanol.

The purity of the isolated glycogen is determined using the Nelsons method. The
isolated glycogen is first hydrolyzed into its monomer units by breaking its glycosidic
bonds. These monomer units (glucose) are quantitatively determined using the
Nelsons method of reducing sugar via spectrophotometric techniques. All
monosaccharides are reducing sugars. In contrast, polysaccharides including glycogen
are not reducing sugars because its anomeric carbons are connected through
glycosidic bond.
Nelsons method is based on the absorbance at 510nm of a blue colored complex
between a copper oxidized sugar and arsenomolybdate. The amount of glucose
present in the sample is determined by comparison with a calibration curve obtained
spectrophotometrically.
The purity of the isolated glycogen can be calculated as follows:
% Purity

actual moles glu cos e / mg of glycogen


theoretical moles glu cos e / mg of glycogen

Where actual moles glu cos e moles glu cos e in the hydrolyzed sample
theoretical moles glu cos e

mg glycogen

180 1 mol

1000
162 180 g
mg glycogen

mg of glycogen

To summarize, the experiment aims to:


(1) Isolate

glycogen from chicken liver through precipitation.

(2) Determine

the percent purity of the isolated glycogen using the Nelson method

of reducing sugars.

Methodology

A. Glycogen Isolation
Fresh chicken liver was obtained. For about two grams of chicken liver, 7.20mL
of hot 30% aqueous KOH was added. The mixture was heated in a boiling water bath
until the liver sample was completely digested. After heating, the mixture was diluted
with 15mL of distilled water and was transferred into 250mL erlenmeyer flask. The
flask was added with 30mL of 95% ethanol and was swirled. The mixture was
decanted so that the precipitate is left in the flask. It was then placed in an ice bath for
20 minutes for complete precipitation. Afterwards, the contents of the flask were
transferred to a 15mL test tube and was centrifuged (7 min, half the maximum speed)
until the precipitate settled down at the bottom of the tube. The supernatant was
discarded and the precipitate was dissolved in 6ml of cold 10% TCA solution. The
resulting was again centrifuged until the brown residue settled at the bottom of the
tube. The supernatant was retained and was added with 15ml of 95% ethanol to
precipitate out the glycogen. It was cooled in an ice bath for 10 minutes and
afterwards was centrifuged. The precipitate was collected in a pre-weighed filter
paper and was washed with 4ml of diethyl ether. The filter paper which contained the
isolated glycogen was weighed the three days after.
B. Glycogen Purity Determination
a. Preparation of a Standard Curve
Table 1 summarizes the preparation of each standards. It should be noted that
after the addition of Nelsons reagent, the tubes were covered with marbles and
placed in a boiling water bath for 20 minutes and was cooled to room
temperature. After the addition of arsenmolybdate, the contents of the tubes were
mixed and was allowed to stand for 5 minutes. Lastly, after the addition of
distilled water, the contents of the samples were again mixed.

The absorbance of the standards were read at 510nm. A standard curve was
made by plotting absorbance against the glucose concentration in mol/ml.
Table 1. Preparation of standard
Nelsons method.
Reagents
Test tube no.
added (ml)
1
2
Glucose
0.00
0.10
(().5mM)
Distilled
1.00
0.90
water
Nelsons
1.00
1.00
reagent
Arsenomoly 1.00
1.00
bdate
reagent
Distilled
7.00
7.00
water

solutions for reducing sugar determination by the


3
0.20

4
0.40

5
0.60

6
0.80

7
1.00

0.80

0.60

0.40

0.20

0.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

7.00

7.00

7.00

7.00

7.00

b. Acid Hydrolysis of Glycogen


In a 50ml beaker, 50mg of glycogen isolate was dissolved in 5.00 ml distilled
water(10mg/ml). Table 2 shows the summary of the protocol followed in the acid
hydrolysis of glycogen. It should be noted that after the addition of 1.2N NaOH
solution, the test tubes were covered with marbles. Test tube 1 and 2 were
allowed to stand at room temperature. Tess tubes 3-8 were placed in a boiling
water bath. From test tube 3, the tubes were removed from the boiling water bath
at a four minute interval. After heating, the reaction for test tubes 3-8 were
terminated by adding 1.00ml of 1.2N NaOH after the specified heating time.
From each diluted samples, 0.50ml aliqout were obtained and diluted to a
final volume of 1.00ml using distilled water. Each solution was assayed with
Nelsons method as indicated in the protocol in table 1. The absorbance was read
at 510nm.
Table 2. Acid Hydrolysis of Glycogen
Reagents
Test tube no.
added (ml)
1
2
3

Distilled
water
Glycogen
solution
(10mg/ml)
2N
HCl
solution
1.2N NaOH
solution
Heating time
(min)
Distilled
water

0.40

0.60

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.40

0.40

0.40

0.40

0.40

0.40

0.40

0.60

0.00

0.60

0.60

0.60

0.60

0.60

0.60

1.00

1.00

0.00

0.00

0.00

0.00

0.00

0.00

10

15

20

25

45

8.00

8.00

8.00

8.00

8.00

8.00

8.00

8.00

References
Seager, S. L.; Slabaugh, M. R. Organic and biochemistry for today; Brooks/Cole:
Pacific Grove, CA, 2000.
Champe, P. C.; Harvey, R. A.; Ferrier, D. R. Biochemistry; Wolters Kluwer
Health/Lippincott Williams & Wilkins: Philadelphia, 2008.
Polysaccharides Definition, List, Functions, Food Examples. (2016). Retrieved
October 09, 2016, from http://www.nutrientsreview.com/carbs/polysaccharides.html
BeMiller, J. M. (2011). Glycogen. In Encyclopedia of Polymer Science and
Technology. John Wiley & Sons.

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