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BIOCHEM 1 L Experiment # 05 2A BC Group 2

ISOLATION, CHARACTERIZATION, AND HYDROLYSIS OF GLYCOGEN AND FROM CHICKEN LIVER


Aquino, Ma. Graciela I.
Department of Biochemistry, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT

Glycogen is the principal storage form of carbohydrate in the mammalian body, which is mainly present
in liver and muscles. This experiment is composed of three parts: extraction, characterization, and
hydrolysis of glycogen. Extraction was done by heat denaturation of glycogen from chicken liver and
addition of 0.1% acetic acid to improve precipitation. After extraction, 95% ethanol was used to precipitate
and purify glycogen, which was seen as white precipitate. The second part involves the general tests for
glycogen which includes Molischs Test and I2 Reaction. Positive results were seen on Molischs Test and
negative in I2 Reaction which are used to test for carbohydrates and starch respectively. The glycogen
extract produced purple interface in the Molischs Test. For the I 2 reaction, a deep red color was expected,
but there was no color change in the solution before, during and after heating. The last part of the
experiment is hydrolysis of glycogen which is subdivided into acidic and enzymatic hydrolysis.
Concentrated HCl was used in acidic hydrolysis while saliva was used in enzymatic hydrolysis. The acidic
and enzymatic hydrolysates were subjected into Benedicts Test and yielded negative results, which
indicates that glycogen is a not a reducing sugar.

INTRODUCTION
Like other polysaccharides, glycogencan also
Glycogen is synthesized from glucose by the undergo hydrolysis. During the reaction, the
pathway of glycogenesis, which occurs mainly in glucose monomer units of glycogen are separated.
liver and muscle. It is the major glucose storage This is being done by the introduction of water in
polymer in animals. It has a highly-branched the glycogen molecule with the presence of strong
structure with linear chains connected by (1-4) acid or base which is summarized in Figure 2 or it
glycosidic bonds and branched points (1-6) every can also be due to the presence of enzymes. [3]
1 in 10 glucose units. It allows the immediate Amylase, an enzyme present in saliva,
release of glucose. [1] catalyzes the hydrolysis of the glycosidic linkages
in starch.

Salivary -amylase (1,4--D-glucan glucano


hydrolase), a monomeric calcium-binding
glycoprotein is involved in preliminary carbohydrate
digestion. It catalyzes the hydrolysis of internal
,14 glycosidic bonds present, yielding a mixture
Figure 1. Branched structure of glycogen of maltose, glucose, oligosaccharides with varying
lengths which constitute branched
Enzymes are proteins that act as catalysts for oligosaccharides. The -glycosidic bond is very
metabolic reactions. They increase the rate of the stable, having a spontaneous rate of hydrolysis of
reaction, but do not influence the kind or amount of 2 10-15 s-1 at room temperature. -Amylase
products formed. In general, each metabolic enhances this rate so enormously, that it can be
reaction has to be catalyzed in the living organism considered as belonging to the most-efficient
by its own special enzyme. [2] enzymes known, increasing the rate 1015-fold. [4]

In this experiment, glycogen was extracted in


chicken liver. General tests were performed to the
glycogen extract specifically Molischs Test and
Iodine Reaction. Glycogen was precipitated using
ethanol. For the hydrolysis, glycogen extract was
hydrolyzed by strong acid and salivary enzyme to
give an estimate of the polysaccharide content of
Figure 2. Hydrolysis of glycogen into glucose the sample.
2

METHODOLOGY C. I2 Reaction
Few drops of 0.01 M I 2 was added into the
I. Extraction of Glycogen from Chicken Liver glycogen extract. The mixture was warmed in a
water bath and cooled after. The color of the
An amount of 13 g of chicken liver was solution was observed before, during, and after
homogenized by using a blender. Boiling water heating of the mixture.
approximately 50 mL was poured into the
homogenized chicken liver. To precipitate the III. Hydrolysis of Glycogen
proteins, the mixture was heated in boiling water
bath for 30 minutes. To improve the precipitation, 1 A. Acid Hydrolysis
mL of 0.1% acetic acid was added. The mixture
was filtered and glycogen extract was obtained, In a test tube, 5 mL of glycogen extract, and 5
which will be used throughout the experiment. drops of conc. HCl was added. The mixture was
covered with marble and boiled in a water bath for
30 minutes. The acid hydrolysate was put in a
refrigerator for Benedicts Test on the next meeting.

B. Enzymatic Hydrolysis

Collection of Saliva

Saliva was collected by rinsing the mouth with


warm distilled water for a minute and the washings
was put in a beaker.
Figure 3. Chicken liver in a blender
Preparation of Dialyzing Bag

Collodion solution was poured into a clean and


dry hard glass (ignition) tube. With the tube in a
horizontal position, the inside was completely
coated by slowly rotating it while pouring off the
excess collodion solution back into its container.
The ignition tube was suspended so the inner
coating of collodion solution will dry. When dried,
the coat was loosened from inside and the
membrane was slowly peeled.
Figure 4. Heating of homogenized chicken liver
In a beaker, 10 mL of glycogen extract and 2.3
mL of saliva was added. The solution was stand at
II. Glycogen Precipitation by Ethanol and General room temperature for 30 minutes and viscosity was
Tests for Glycogen noted. The solution was introduced in a dialyzing
bag and suspended overnight in a small flask with
In each test, 1 mL of glycogen extract was 50 mL distilled water. In the next meeting, the
added into a test tube. solution was removed and the dialyzing bag was
discarded. The solution inside the flask was
A. Ethanol Precipitation concentrated to a volume of 10 mL using an open
flame. Presence of reducing sugars was tested by
An amount of 10 drops was added into the performing Benedicts Test in the hydrolysis.
glycogen extract to induce precipitation.
Benedicts Test
B. Molischs Test
In two separate test tubes, 5 drops of acidic
Few drops of Molischs reagent was added into hydrolysate and 5 drops of enzymatic hydrolysate
the glycogen extract. An amount of 2 mL conc. were added respectively. An amount of 1 mL of
H2SO4 was carefully added to the side of the test Benedicts soution was added to each hydrolysate.
tube to form a layer. The mixtures were heated in a boiling water bath at
3
the same time. After heating, the result was dehydrates hexoses to form 5-hydroxymethyl
observed. furfural in Figure 8. The furfurals further react with
-naphthol present in the test reagent as can be
RESULTS AND DISCUSSION seen from Figure 9 to produce a purple product
which is shown in Figure 10. [6]
I. Extraction of Glycogen from Chicken Liver

Figure 7. Dehydration of pentoses to form furfural

Figure 5. Glycogen extract

Glycogen was successfully isolated. Figure 5


shows the isolated glycogen which is a yellow
solution with small, white precipitate.
The precipitation of the proteins was done by
boiling the solution. During heating, glycogen was Figure 8. Dehydration of hexoses to form 5-
left soluble in the solution while proteins were hydroxymethyl furfural
denatured and precipitated. The precipitation
process was enhanced by the addition of 0.1%
acetic acid, The impurities or precipitate was
separated from the solution by the use of gravity
filtration.

II. Glycogen Precipitation by Ethanol and General


Tests for Glycogen
Figure 9. Further reaction of furfurals with -
A. Ethanol Precipitation naphthol

Figure 6. Glycogen precipitation by ethanol Figure 10. Formation of purple interface

Glycogen is a polymer which is used to trap C. I2 Reaction


the nucleic acids. In ethanol, glycogen is in soluble
so it forms polymer structure which can be seen as The use of iodine is useful to distinguish starch
white precipitate. [5] Precipitation is induced by the and glycogen from other polysaccharides. Iodine
loss of water shell of glycogen molecules. yields a blue-black color in the presence of starch
while glycogen complexes with iodine to give a
B. Molischs Test deep red color. Other polysaccharides and
monosaccharides yield no color change; the test
solution remains the characteristic brown-yellow of
Molischs Test shows positive test for all
the reagent. Glycogen forms helical coils. Iodine
carbohydrates. The test reagent dehydrates
atoms can then fit into the helices to form a
pentoses to form furfural in Figure 7 and
4
glycogen-iodine complex. Starch in the form of the glucose derivative, the result will be a
amylose and amylopectin has less branches than tetramethyl ether of the pyranose hemiacetal. This
glycogen. This means that the helices of starch are compound will, of course, undergo typical aldehyde
longer than glycogen, therefore binding more reactions. [9]
iodine atoms. The result is that the color produced
by a starch-iodine complex is more intense than
that obtained with a glycogen-iodine complex. [7]
In the experiment, the color of glycogen-iodine
mixture was observed before, during, and after
heating. As shown in Figure 11, white precipitate
was formed. After heating, still no change in color
of the mixture was observed.
The shade of the glycogen complex is
characteristic, that it can be recognized outwardly
when the grouping of the iodine is as low as
0.00002 M at 20 C. The shading affectability Figure 11. Acid hydrolysate of glycogen
reduces with expanding temperature (ten times
less delicate at 50 C), and upon the expansion of B. Enzymatic Hydrolysis
natural solvents, for example, ethanol. [8]
Negative results may be due to impurities or Enzyme-catalyzed hydrolyses are more
improper preparation of glycogen extract and specific with respect to bonds cleaved, for
iodine reagent. example, -amylase of human saliva. The -
amylase catalyzes the rapid, random hydrolysis of
internal -1,4 bonds. They do not however,
hydrolyze -1,6 linkages, regardless of molecular
size, nor do they hydrolyze maltose. Thus,
glycogen is initially split by -amylase action into
branched dextrins of medium molecular weight and
only small amounts if maltose is formed. The final
degradation products of the action of -amylase on
glycogen are glucose, maltose and isomaltose. The
glucose is formed by the relatively slow end
cleavages of the oligosaccharides. [10]
Figure 10. Heating of glycogen-iodine solution Enzymatic hydrolysis was done by the process
of dialysis, which includes a semi-permeable
III. Hydrolysis of Glycogen membrane that allows molecules to pass through
via diffusion into the surrounding medium.
A. Acid Hydrolysis
Glycogen is a polymer of glucose. This is In the experiment, the dialyzing bag, which is a
easily demonstrated by acid-catalyzed hydrolysis to collodion solution composed of pyroxylin film, ether,
the monosaccharide. The acid hydrolysis is and alcohol, served as the membrane that allows
addition of H+/H2O to a covalent bond. In the case monosaccharides and disaccharides to pass
of glycogen, the glycosidic covalent bonds are the through into the distilled water medium. The sugar
target of acid hydrolysis. Heating of glycogen in the solution produced by addition of salivary enzyme
presence of conc. HCl causes its hydrolysis into into the glycogen extract was more viscous before
glucose because of the free aldehyde group, hydrolysis. After an hour, the solution became less
making glycogen a strongly reducing viscous as shown in Figure 12.
monosaccharide. These glycosidic linkages (1-4
and 1-6 carbons) are joining the monosaccharide in
glycogen and their hydrolysis is quite random.
Many oligosaccharides form in between as
intermediates eventually result as glucose. The
reaction is shown as:

C12H22O11+H+/H2O-------->2(C6H12O6)

Acid hydrolysis of acetals regenerates the


carbonyl and alcohol components, in the case of
5
Figure 12. Enzymatic hydrolysate of glycogen The Benedicts Test both for the acid and
enzymatic hydrolysate gave a false result since
glycogen is a non-reducing sugar.

Benedicts Test CONCLUSION


The Benedict's Test is used detect the

[7] Chemistry Laboratory. (n.d.). Retrieved May 01, 2017, from


http://generalchemistrylab.blogspot.com/2011/12/iodine-test-for-starch-and-glycogen.html
[8] A. (2016, October 02). Iodine test for Starch- Its Principle, Reagents, Procedure etc. Retrieved May 01,
2017, from http://allmedtests.com/iodine-test-starch/
[9] Starch Hydrolysis by Amylase. (n.d.). Retrieved May 01, 2017, from
http://eng.umd.edu/~nsw/ench485/lab5.html
[10] Starch Hydrolysis by Amylase. (n.d.). Retrieved May 01, 2017, from
http://eng.umd.edu/~nsw/ench485/lab5.html
presence of reducing sugars (sugars with a free REFERENCES
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precipitate. The reaction is as follows: ps/chm/100/dgodambe/thedisk/carbo/molisch/molis
In this experiment, heat denaturation is the principle involved in extraction of glycogen. Addition of
ethanol results in glycogen precipitation and allows to obtain relatively purified glycogen.
The general tests for glycogen involves Molischs Test and I2 Reaction. Molischs Test which is used for
detection of carbohydrates, involves the principle of hydrolysis, dehydration, and condensation with -
naphthol. The positive result of purple interface was obtained. For I 2 Reaction, negative result was
obtained because of possible presence of impurities in the glycogen extract or contamination of the iodine
reagent.
Glycosidic bonds in glycogen are resistant to hydrolytic activity of OH - at elevated temperature, which
allows only the hydrolysis of glycogen by acid and salivary enzyme. Dialysis during enzymatic hydrolysis
enables monosaccharides and disaccharides to diffuse into the water medium.
Benedicts Test for reducing sugars involves the principle of oxidation in the less basic medium.
Enzymatic and acidic hydrolysate yielded negative results which indicates that glycogen is a non-reducing
sugar.

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R-CHO + 2Cu +5OH --------> R-COO +Cu2O +
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https://www.musclesound.com/what-is-glycogen/
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