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EXPERIMENT 6: ISOLATION OF GLYCOGEN

2011-35493, 2011-85007
Biochem 34.1 HEJ, Sir Marvin Pelovello

I. ABSTRACT
Glycogen is a vital carbohydrate that functions as the storage form of energy found in animal cells. This experiment
generally aims to learn the techniques and understand the principles in isolating glycogen. Specifically, its purpose
is to explain the principle behind using cold precipitation for isolating glycogen and to confirm the presence of
carbohydrates using qualitative tests. Crude glycogen was efficiently purified and extracted from chicken liver by
performing methods of homogenization, centrifugation, and cold precipitation by ethanol. Neutralized hydrolyzate
was then produced from half of the extracted crude sample through acid hydrolysis with heat. The basis of the
Molisch test is the condensation reaction between furfural compound and -naphthol wherein a purple ring
formation that was observed in the samples confirms the presence of carbohydrates. The hydrolyzate theoretically
gives a positive result, formation of red precipitate, for the detection of reducing sugars by Benedicts test since
ideally its components are unbound glucose which is a reducing sugar. Reaction with phenylhydrazine results to the
formation of osazone crystals or a yellow solution, the positive outcome for the Osazone test, which was obtained,
confirming the presence of glucose. Barfoeds test and Seliwanoffs test can be done to improve characterization of
carbohydrates.

II. KEYWORDS: glycogen, carbohydrates, Molisch test, Benedicts test, Osazone test

III. INTRODUCTION
Carbohydrates are the most abundant principles in isolating glycogen. Specifically, the
biomolecules on earth. Each year, photosynthesis experiment aims to explain the principles behind
converts more than 100 billion metric tons of carbon using cold precipitation for the isolation and to
dioxide and water into cellulose and other plant confirm the presence of carbohydrates using
products. Certain carbohydrates, such as sugar and qualitative tests.
starch, are dietary staples in most parts of the world,
and the oxidation of carbohydrates is the central IV. EXPERIMENTAL
energy-yielding pathway in most non-photosynthetic Isolation of glycogen started with the
cells (Nelson & Cox, 2012). separation of the desired compound from the
supernatant fraction of chicken liver. The chicken
Originally, carbohydrates are referred to as liver was washed and pat dried before obtaining 20
compounds containing Cn(H2O)n. This formula is only grams of the sample. A small volume of 7.4 pH
true for simple sugars, or monosaccharides. Other phosphate buffer was added. The sample was
types of carbohydrates, oligosaccharides and minced finely and placed in the homogenizer with
polysaccharides, are based on monosaccharide units 150 mL of the homogenizing solution (7.4 pH
and have slightly different general formulas phosphate buffer). The sample was homogenized to
(Campbell & Farrell, 2013). Generally, carbohydrates even and smooth consistency and was then
are macromolecules that are made up of polymers of transferred to falcon tubes. The falcon tubes were
polyhydroxy aldehydes or ketones linked together by centrifuged at 3000 rpm for 10 minutes. The
glycosidic bonds. They are called aldoses or ketoses, precipitate was discarded while 15 mL of the
depending on the nature of the carbonyl group supernatant was collected in a test tube. One mL of
present. They are called trioses, pentoses, depending 10% acetic acid was added to the test tube, covered
on the number of carbons in the molecule. with a marble, and placed in a boiling water bath for
five minutes. The supernatant was transferred to
Isolation techniques for carbohydrates are falcon tubes, cooled, and then centrifuged at 3000
easier to perform due to the weak interactions rpm for five minutes. The precipitate was discarded
involved as compared to other biomolecules. They and the supernatant was transferred to a test tube
are water-soluble and do not denature readily. and cooled to about 10oC in the refrigerator. Absolute
ethanol was added to fill half to a third of the test
The general objective of the experiment is to tube. Appearance of white, flocculent precipitate was
be able to learn the techniques and understand the observed. The solution is placed in the refrigerator for

Biochem 34.1 Isolation of Glycogen Page |1


an hour to ensure precipitation. Then, the solution is present in most of the tissues, however it is stored in
centrifuged at 3000 rpm for 10 minutes. The two major sites which are the liver and muscle
precipitate is collected and washed twice with (skeletal). In the experiment, the preferred source of
distilled water. It was dissolved in five mL water. The glycogen is liver rather than muscle tissue because
sample is labeled as the crude isolate. the concentration of glycogen is higher in the liver
(10% by weight) than in muscle (2% by weight). In the
For the hydrolysis part of the experiment, half liver, regulation of glycogen synthesis and
of the crude isolate was separated and added to the degradation is carried out to maintain the level of
same amount of 6M HCl. The test tube was covered glucose in blood required by the organism (Berg, et
with marble and placed in a boiling water bath for 30 al., 2012).
minutes. The test tube was cooled and neutralized
with concentrated ammonium hydroxide. The sample Glycogen appears as granules in cells
is labeled NH. specifically in the cytosol ranging in diameter from 10
to 40 nm (Chhabra, 2015). Homogenization using
There are three qualitative tests involved in the blender was done in order to break open these
this experiment. All tests involved two positive cells and disperse their contents in an aqueous
controls, namely 1% glucose and 1% arabinose, and buffer. The phosphate buffer with pH 7.4 was utilized
a negative control with distilled water. to avoid any disintegration of important subcellular
components. At the centrifugation rate of 3000 rpm
The first one is the Molisch test. Ten drops of in 10 minutes duration, higher molecular weight
the crude isolate and NH were placed in different test macromolecules found in the cells like proteins and
tubes. Ten drops of freshly prepared Molisch reagent nucleic acids were isolated as the precipitate. Further
were added. The solution is mixed thoroughly. The purification of the supernatant was done by adding
test tube is tilted carefully while one mL of H2SO4 was 10% HOAc. Addition of acetic acid promotes the
allowed to slide down the side of the test tube to denaturation of residual proteins that consequently
form a layer at the bottom. The color at the interface causes precipitation. Then, subjecting the solution in
was observed. heat affects hydrogen bonding and non-polar
hydrophobic interactions that are present, effectively
The second test is the Benedicts test. Ten separating any unwanted components during the
drops of the crude isolate and NH were placed in subsequent centrifugation at 3000 rpm for 5
different test tube. Five drops of the Benedicts minutes (Nelson & Cox, 2013).
reagent were added. The test tubes were covered
with marble and placed in a boiling water bath for
five minutes. Changes in the appearance of the
solution were noted.

Finally, for the Osazone test, five drops of the


crude isolate and NH were placed in different test
tubes. Ten drops of freshly prepared phenylhydrazine
reagent were added. The test tubes were covered
with marble and placed in a boiling water bath for
five minutes. First appearance of yellow crystals was
Figure 1. Schematic two-dimensional cross-sectional view of
timed. The test tubes are then allowed to cool to
glycogen. Image retrieved from
room temperature, and then a few drops of the https://en.wikipedia.org/wiki/Glycogen.
solutions are placed on separate glass slides and
viewed under the microscope. The crystals formed Glycogen is soluble in water due to its globular
are visible. structure wherein the hydrophilic hydroxyl groups are
placed outside the mesh cells (see Figure 1) making
V. RESULTS AND DISCUSSION them available for water interaction. However, it is
The crude sample of glycogen extracted from insoluble in alcohol. Hence, introduction of absolute
chicken liver tissue is observed to be white and ethanol precipitates glycogen in the solution and
cloudy. Glycogen is a polysaccharide that serves as placing it under low temperature completes the
the fuel-storage form of glucose in animal cells. It is reaction (azaquar, 2011). Successful extraction of

Biochem 34.1 Isolation of Glycogen Page |2


glycogen is achieved upon the formation of
precipitates. Further analyses through performing
hydrolysis then qualitative tests are done to validate
the product.

Glycogen can either undergo chemical


hydrolysis or enzymatic hydrolysis. Chemical
hydrolysis is done in the experiment by adding 6 M
HCl to the crude sample of glycogen. With the
introduction of water in the presence of strong
aqueous acid, hydrolysis breaks the glycosidic bonds, Figure 3. Theoretical positive result for carbohydrates for Molisch
though fairly stable, and frees monomeric units of test. (Chhabra, 2014)

glycogen which is glucose (Glycogen, 2013). The


reaction is illustrated in Figure 2 below. Placing the The first qualitative test that was done is the
solution in a boiling water bath facilitates complete general test for carbohydrates, the Molisch test. This
hydrolysis of the sample. In addition, heating and test is based on a two-step analysis. Figure 4 below
adding strong acid causes for the liberated shows the simplified mechanism of the Molisch test.
monosaccharides to be dehydrated and produces First is the production of an aldehyde, either furfural
furfural derivatives. Glucose, upon addition of strong or its derivatives, produced by the dehydration of a
acids, yields 5-hydroxymethyl furfural (Galewski, et monosaccharide upon in contact with a concentrated
al., 2013). Concentrated NH4OH is then added to strong acid like H2SO4. In the experiment, the acid is
neutralize the sample after being subjected to acid. gradually slid down the walls of the test tube so that
sublayering is effectively achieved (for positive
results). Pentose (five-carbon monosaccharide) and
hexose (six-carbon monosaccharide) like glucose
forms furfural and hydroxymethyl furfural
respectively. Next is the reaction with the Molisch
reagent. Furfural compound specifically
hydroxymethyl furfural is very reactive and condenses
with phenolic compounds such as -naphthol
Figure 2. Chemical hydrolysis at the (14) glycosidic bond of (Molisch reagent) to form colored products. The
glycogen forming glucose units. Image retrieved from positive outcome is a reddish violet or purple colored
https://www.boundless.com/physiology/textbooks/boundless- ring at the interface of two liquids (Nigam & Ayyagari,
anatomy-and-physiology-textbook/digestive-system-23/chemical-
2007).
digestion-224/mechanisms-of-chemical-digestion-1103-
8914/images/hydrolysis-by-amylase.

Three qualitative tests were done to


characterize glycogen and confirm the presence of its
components. They are Molisch test, Benedicts test,
and Osazone test. Note that the tests werent
completely performed on other test compounds. So,
Figure 4. Reaction mechanism of Molisch test (of D-glucose).
some of the results shown are theoretical. (Nigam & Ayyagari, 2007)

Table 1. Visible results obtained in performing Based on Table 1, positive results are given by
Molisch test on crude sample, NH sample, (+) 1% the crude and neutralized hydrolyzate test
glucose, (-) distilled H2O. (Asterisk mark, *, indicates compounds. This confirms that the extracted crude
theoretical result) sample is a carbohydrate, and the hydrolyzed state is
Test Compounds composed of carbohydrates. However, this test
Molisch
(+) 1% (-) distilled doesnt really confirm if the isolated carbohydrate is
Test crude NH
glucose H2O glycogen because this test is positive for all types of
Visible Purple Purple Purple No carbohydrates. Moreover, this is nonspecific for
result ring solution ring* discoloration* carbohydrates since this will give a positive for
glycoproteins, glycolipids, and nucleic acids as well.

Biochem 34.1 Isolation of Glycogen Page |3


Table 2. Visible results obtained in performing 2NaOH + CuSO4 Cu(OH)2 + Na2SO4
Benedicts test on crude sample, NH sample, (+) 1% Cu(OH)2 + Na citrate Cu(OH)2:Na citrate complex
glucose, (-) distilled H2O. (Asterisk mark, *, indicates Cu(OH)2 CuO + H2O
theoretical result)
D-glucose + 2CuO D-gluconic acid + Cu2O
Test Compounds
Benedicts (-) Sodium carbonate is responsible for the
(+) 1%
Test crude NH distilled alkaline environment of the solution by providing OH-
glucose
H2O ions whereas copper sulfate is the source for cupric
Red (II) ions. The sodium citrate compound is a chelating
Visible Blue Blue Blue
solution or
result solution solution solution* agent for metallic ion forming a complex. This
precipitate*
ensures that the cupric ions are retained in the
solution. The final reaction is the reduction of copper
(II) oxide to a colored precipitate of copper (I) oxide
while the sugar, in this case is D-glucose, is oxidized
to a sugar acid, D-gluconic acid. This reaction is
facilitated by heat. The formation of the cuprous
oxide precipitate indicates a positive result. Based on
concentration (in g %) of reducing sugars present in
the sample, the color of the precipitate or solution
varies from green (0.1-0.5 g %), yellow (0.5-1.0 g %),
Figure 5. Theoretical results for carbohydrates in Benedicts test. orange (1.0-1.5 g %), red (1.5-2.0 g %), brick-red
Negative result is shown in the first tube. The last three tubes (>2.0 g %). This makes Benedicts test a semi-
show positive results (color depends on concentration of reducing
sugar). (Aryal, 2015)
quantitative test (IMDCBiochem, 2010).

Benedicts test is specific and highly sensitive The obtained result (see Table 2) for the crude
to reducing sugars. This makes it useful to sample under Benedicts test corresponds to the
distinguish between reducing and non-reducing negative control. This confirms that glycogen is a
sugars. Reducing sugars are able to reduce solutions non-reducing sugar despite having a reducing end.
of various metallic ions. The general principle behind The presence of a reducing end may not be sufficient
the Benedicts test is that in weak alkaline solution to be detected by the test. For the hydrolyzate, it is a
aided with heat, the reducing sugars reduce cupric negative outcome incorrect result. Theoretical
(II) ions to green/yellow/orange/red precipitate of result for the hydrolyzate shows a positive result
cuprous (I) ions, while the sugars themselves are since glucose which is a reducing sugar is present.
oxidized to sugar acids. Oxidation of reducing sugars Possible cause for the incorrect result is the
is done in the free aldehyde functional groups of incomplete release of glucose monomers from the
aldoses (glucose, mannose, etc.). This test also glycogen.
detects if the aldehyde group in the sugar is unbound
(free) or bound. On the other hand, oxidation is also Table 3. Visible results observed and time of crystal
possible for ketoses, sugars with ketone functional appearance recorded in performing Osazone test on
group. Fructose, a ketose with -hydroxymethyl crude sample, NH sample, (+) 1% glucose, (+) 1%
ketone group, gives a positive result for Benedicts arabinose, (-) distilled H2O. (Asterisk mark, *,
test. This is due to the fact that under high pH indicates theoretical result)
(alkaline), fructose is converted to isomers of glucose Test compounds
Osazone (-)
and mannose which are aldoses and thus exhibits (+) 1% (+) 1%
Test crude NH distilled
reducing properties (Garcia, et al., n.d.). arabinose
glucose H2O
Visible Yellow Yellow Yellow Yellow Clear
Benedicts reagent is composed of CuSO4,
result solution* solution solution* solution solution*
sodium carbonate, and sodium citrate. These 4 mins 1 min
compounds in the presence of heat and reducing Time of 4-5 10
& 30 & 15 -
sugars carry out the following reactions.
appearance
secs secs
mins* mins*

Na2CO3 + 2H2O 2NaOH + H2CO3

Biochem 34.1 Isolation of Glycogen Page |4


Figure 6. Theoretical positive results of carbohydrates in Osazone Figure 7. Reaction mechanism of Osazone test. (Nigam & Ayyagari,
test. (From L to R: glucose, fructose, sucrose). (Chhabra, 2015) 2007)

Osazone test is the last qualitative test After performing the Osazone test, the
performed in the experiment. It is named as such neutralized hydrolyzate was observed as a yellow-
since this detects and identifies reducing sugars like colored solution (prefer to Table 3). This is similar to
monosaccharides and disaccharides based on the the obtained result for positive controls- 1%
formation of osazone and its formation time. Sugar arabinose, 1% glucose (theoretical). This confirms
osazones are yellow crystalline compounds the presence of reducing sugar which is glucose in
characteristic to every reducing sugars, therefore are the sample. It can be assumed that hydrolysis of
seen in various shapes and forms (under the glycogen was successful and release of glucose as
microscope). Formation of these crystals under monomeric units was effectively executed. It is
certain conditions, either hot or cold, further observed that the time of appearance of osazone
identifies the reducing sugar. Generally, crystals for the crude sample is within the theoretical
monosaccharides give crystals on heating and all range of the positive control (1% glucose) which
disaccharides give crystal on cooling (IMDCBiochem, verifies that glucose is present in glycogen. The time
2010). In addition, the crystal formation time of appearance under NH is theoretically similar to the
determines the reducing sugar in the sample. For 1% glucose positive control since it is expected that
instance, glucosazone is formed by glucose within 4- the reducing sugar in the hydrolyzate is ideally
5 minutes of heating. glucose.

The reagent used in this test is It is to be noted that the samples were not
phenylhydrazine reagent that is made up of viewed under the microscope, thus there are no are
phenylhydrazine and sodium acetate diluted in water. images procured in the experiment for Osazone test.
Sodium acetate provides a constant pH in the Instead, theoretical image is shown below displaying
solution. The mechanism of this test (see Figure 7) the crystal (glucosazone) formed by glucose in
includes the reaction of carbonyl group of the Osazone test.
reducing carbohydrate with phenylhydrazine under
boiling temperature forming phenylhydrazone. Then,
this resulting product reacts with another two
molecules of phenylhydrazine producing the
insoluble osazone crystals. Formation of these
osazone crystals suggests a positive result for
Osazone test (Nigam & Ayyagari, 2007). False
negative results will be produced if the added
phenylhydrazine reagent is insufficient or the heat is
not at boiling temperature causing for an incomplete
reaction to occur. Figure 8. Needle- shaped crystals of glucosazone viewed under the
microscope. (Chhabra, 2014)

Fructose and mannose will analogously form


needle-shaped osazone crystals, fructosazone and
mannosazone respectively. Hexoses, when reacted
with phenylhydrazine, only involve the carbons at C1
and C2 positions. Glucose, fructose, and mannose

Biochem 34.1 Isolation of Glycogen Page |5


mainly differ in their configuration or the functional sugar. The latter test distinguishes reducing sugars
group at the C1 and C2 positions. Thus, the among each other basing on the osazone crystal
differences in these carbon positions do not affect formation upon reaction with phenylhydrazine
the shapes of the crystals formed (Nigam & Ayyagari, reagent. Positive outcome is given by a yellow
2007). solution or yellow crystals (viewed under
microscope). The procured positive result for NH,
neutralized hydrolyzate, confirms the presence of
reducing sugar, ideally glucose. The time of
appearance of osazone by the crude sample is within
the theoretical range of osazone formation time for
1% glucose that boosts the confidence that the
extracted sample is glycogen.
Figure 9. Reaction illustrating the formation of similar osazone
from D-(+)-glucose and D-(+)-mannose. (Chhabra, 2015)
It is recommended that other qualitative tests
VI. CONCLUSIONS AND RECOMMENDATIONS are performed to improve the analysis of the sample
Purification and isolation of glycogen from and its components. Additional tests may include
chicken liver tissue was effectively achieved by Barfoeds test, Seliwanoffs test, Bials-Orcinol test,
performing homogenization, centrifugation, and and Mucic acid test. The reagents utilized should be
precipitation (use of acetic acid and cold ethanol). properly stored to avoid degradation and they should
Further validation was done by performing three be frequently updated so that better results are
qualitative tests. The tests include Molisch test, achieved. Proper adherence to the given procedure is
Benedicts test, and Osazone test. suggested to avoid any erroneous outcomes and to
avoid accidents as well.
Molisch test is a general test for
carbohydrates. This is based on the reaction of - VII. REFERENCES
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