2011-35493, 2011-85007 Biochem 34.1 HEJ, Sir Marvin Pelovello
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 α-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 Benedict’s 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. Barfoed’s test and Seliwanoff’s test can be done to improve characterization of carbohydrates. glycogen, carbohydrates,
Carbohydrates
are
the
most
Molisch test, Benedict’s test, Osazone test
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 dioxide and water into cellulose and other plant
using cold precipitation for the isolation and to confirm the presence of carbohydrates using
products. Certain carbohydrates, such as sugar and starch, are dietary staples in most parts of the world, and the oxidation of carbohydrates is the central
qualitative tests.
energy-yielding pathway in most non-photosynthetic cells (Nelson & Cox, 2012).
Isolation of glycogen started with the separation of the desired compound from the supernatant fraction of chicken liver. The chicken
Originally, carbohydrates are referred to as compounds containing Cn(H2O)n. This formula is only true for simple sugars, or monosaccharides. Other
liver was washed and pat dried before obtaining 20 grams of the sample. A small volume of 7.4 pH phosphate buffer was added. The sample was
types of carbohydrates, oligosaccharides and polysaccharides, are based on monosaccharide units and have slightly different general formulas
minced finely and placed in the homogenizer with 150 mL of the homogenizing solution (7.4 pH phosphate buffer). The sample was homogenized to
(Campbell & Farrell, 2013). Generally, carbohydrates
even
are macromolecules that are made up of polymers of polyhydroxy aldehydes or ketones linked together by
transferred to falcon tubes. The falcon tubes were centrifuged at 3000 rpm for 10 minutes. The
glycosidic bonds. They are called aldoses or ketoses, depending on the nature of the carbonyl group present. They are called trioses, pentoses, depending on the number of carbons in the molecule.
precipitate was discarded while 15 mL of the supernatant was collected in a test tube. One mL of 10% acetic acid was added to the test tube, covered with a marble, and placed in a boiling water bath for five minutes. The supernatant was transferred to
Isolation techniques for carbohydrates are easier to perform due to the weak interactions involved as compared to other biomolecules. They
falcon tubes, cooled, and then centrifuged at 3000 rpm for five minutes. The precipitate was discarded and the supernatant was transferred to a test tube
are water-soluble and do not denature readily.
and cooled to about 10 oC in the refrigerator. Absolute ethanol was added to fill half to a third of the test tube. Appearance of white, flocculent precipitate was observed. The solution is placed in the refrigerator for
The general objective of the experiment is to be able to learn the techniques and understand the
Biochem 34.1 │ Isolation of Glycogen
and
smooth
consistency
and
was
then
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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 precipitate is collected and washed twice with distilled water. It was dissolved in five mL water. The
two major sites which are the liver and muscle (skeletal). In the experiment, the preferred source of glycogen is liver rather than muscle tissue because
sample is labeled as the crude isolate. For the hydrolysis part of the experiment, half
the concentration of glycogen is higher in the liver (10% by weight) than in muscle (2% by weight). In the liver, regulation of glycogen synthesis and
of the crude isolate was separated and added to the same amount of 6M HCl. The test tube was covered
degradation is carried out to maintain the level of glucose in blood required by the organism (Berg, et
with marble and placed in a boiling water bath for 30 minutes. The test tube was cooled and neutralized with concentrated ammonium hydroxide. The sample
al., 2012).
is labeled NH. There are three qualitative tests involved in this experiment. All tests involved two positive
specifically in the cytosol ranging in diameter from 10 to 40 nm (Chhabra, 2015). Homogenization using the blender was done in order to break open these cells and disperse their contents in an aqueous
controls, namely 1% glucose and 1% arabinose, and a negative control with distilled water.
buffer. The phosphate buffer with pH 7.4 was utilized to avoid any disintegration of important subcellular
The first one is the Molisch test. Ten drops of the crude isolate and NH were placed in different test
components. At the centrifugation rate of 3000 rpm in 10 minutes duration, higher molecular weight macromolecules found in the cells like proteins and
tubes. Ten drops of freshly prepared Molisch reagent were added. The solution is mixed thoroughly. The
nucleic acids were isolated as the precipitate. Further 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 form a layer at the bottom. The color at the interface
denaturation of residual proteins that consequently causes precipitation. Then, subjecting the solution in
was observed.
heat affects hydrogen bonding and non-polar hydrophobic interactions that are present, effectively separating any unwanted components during the
The second test is the Benedict’s test. Ten drops of the crude isolate and NH were placed in
different test tube. Five drops of the Benedict’s
Glycogen
appears
as
granules
in
cells
subsequent centrifugation at 3000 rpm for 5 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 timed. The test tubes are then allowed to cool to room temperature, and then a few drops of the solutions are placed on separate glass slides and viewed under the microscope. The crystals formed are visible.
The crude sample of glycogen extracted from chicken liver tissue is observed to be white and cloudy. Glycogen is a polysaccharide that serves as the fuel-storage form of glucose in animal cells. It is
Biochem 34.1 │ Isolation of Glycogen
Schematic two-dimensional cross-sectional view of glycogen. Image retrieved from https://en.wikipedia.org/wiki/Glycogen.
Glycogen is soluble in water due to its globular structure wherein the hydrophilic hydroxyl groups are placed outside the mesh cells (see Figure 1) making them available for water interaction. However, it is insoluble in alcohol. Hence, introduction of absolute ethanol precipitates glycogen in the solution and placing it under low temperature completes the reaction (azaquar, 2011). Successful extraction of
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glycogen
is
achieved
upon
the
formation
of
precipitates. Further analyses through performing hydrolysis then qualitative tests are done to validate the product. Glycogen hydrolysis or
can either undergo enzymatic hydrolysis.
chemical 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, though fairly stable, and frees monomeric units of glycogen which is glucose (Glycogen, 2013). The reaction is illustrated in Figure 2 below. Placing the solution in a boiling water bath facilitates complete hydrolysis of the sample. In addition, heating and adding strong acid causes for the liberated monosaccharides to be dehydrated and produces furfural derivatives. Glucose, upon addition of strong acids, yields 5-hydroxymethyl furfural (Galewski, et al., 2013). Concentrated NH4OH is then added to neutralize the sample after being subjected to acid.
Theoretical positive result for carbohydrates for Molisch test. (Chhabra, 2014)
The first qualitative test that was done is the general test for carbohydrates, the Molisch test. This test is based on a two-step analysis. Figure 4 below shows the simplified mechanism of the Molisch test. First is the production of an aldehyde, either furfural or its derivatives, produced by the dehydration of a monosaccharide upon in contact with a concentrated strong acid like H2SO4. In the experiment, the acid is 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 a nd condenses
with phenolic compounds such as α-naphthol Chemical hydrolysis at the (α1→4) glycosidic bond of glycogen forming glucose units. Image retrieved from https://www.boundless.com/physiology/textbooks/boundlessanatomy-and-physiology-textbook/digestive-system-23/chemicaldigestion-224/mechanisms-of-chemical-digestion-11038914/images/hydrolysis-by-amylase.
(Molisch reagent) to form colored products. The positive outcome is a reddish violet or purple colored ring at the interface of two liquids (Nigam & Ayyagari, 2007).
Three qualitative tests were done to characterize glycogen and confirm the presence of its
components. They are Molisch test, Benedict’s test, and Osazone test. Note that the tests weren’t completely performed on other test compounds. So, some of the results shown are theoretical. . Visible results obtained in performing Molisch test on crude sample, NH sample, (+) 1% glucose, (-) distilled H2O. (Asterisk mark, *, indicates theoretical result)
Reaction mechanism of Molisch test (of (Nigam & Ayyagari, 2007)
the
D-glucose).
Based on Table 1, positive results are given by crude and neutralized hydrolyzate test
compounds. This confirms that the extracted crude sample is a carbohydrate, and the hydrolyzed state is composed of carbohydrates. However, this test
doesn’t really confirm if the isolated carbohydrate is Purple ring
Purple solution
Purple ring*
Biochem 34.1 │ Isolation of Glycogen
No discoloration*
glycogen because this test is positive for all types of carbohydrates. Moreover, this is nonspecific for carbohydrates since this will give a positive for glycoproteins, glycolipids, and nucleic acids as well.
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Visible results obtained in performing
Benedict’s test on crude sample, NH sample, (+) 1% glucose, (-) distilled H2O. (Asterisk mark, *, indicates theoretical result)
2NaOH + CuSO4 → Cu(OH)2 + Na2SO4 Cu(OH)2 + Na citrate → Cu(OH)2:Na citrate complex Cu(OH)2 → CuO + H 2O D-glucose
+ 2CuO
∆
→ D-gluconic
acid + Cu2O↓
Sodium carbonate is responsible for the alkaline environment of the solution by providing OH Blue solution
Blue solution
Red solution or precipitate*
Blue solution*
ions whereas copper sulfate is the source for cupric (II) ions. The sodium citrate compound is a chelating agent for metallic ion forming a complex. This 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
Theoretical results for carbohydrates in Benedict’s test. Negative result is shown in the first tube. The last three tubes show positive results (color depends on concentration of reducing sugar). (Aryal, 2015)
Benedict’s test is specific and highly sensitive to reducing sugars. This makes it useful to distinguish between reducing and non-reducing sugars. Reducing sugars are able to reduce solutions
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 %), orange (1.0-1.5 g %), red (1.5-2.0 g %), brick-red (>2.0 g %). This makes Benedict’s test quantitative test (IMDCBiochem, 2010).
a semi-
The obtained result (see Table 2) for the crude
sample under Benedict’s test corresponds to the negative control. This confirms that glycogen is a non-reducing sugar despite having a reducing end. The presence of a reducing end may not be sufficient
of various metallic ions. The general principle behind the Benedict’s test is that in weak alkaline solution aided with heat, the reducing sugars reduce cupric (II) ions to green/yellow/orange/red precipitate of cuprous (I) ions, while the sugars themselves are oxidized to sugar acids. Oxidation of reducing sugars
to be detected by the test. For the hydrolyzate, it is a negative outcome – incorrect result. Theoretical result for the hydrolyzate shows a positive result since glucose which is a reducing sugar is present.
is done in the free aldehyde functional groups of 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 possible for ketoses, sugars with ketone functional g roup. roup. Fructose, a ketose with α-hydroxymethyl
ketone group, gives a positive result for Benedict’s test. This is due to the fact that under high pH (alkaline), fructose is converted to isomers of glucose and mannose which are aldoses and thus exhibits reducing properties (Garcia, et al., n.d.).
Possible cause for the incorrect result is the incomplete release of glucose monomers from the
Visible results observed and time of crystal appearance recorded in performing Osazone test on crude sample, NH sample, (+) 1% glucose, (+) 1% arabinose, (-) distilled H2O. (Asterisk mark, *, indicates theoretical result)
crude
NH
(+) 1% glucose
(-) (+) 1% arabinose
distilled H2O
Benedict’s reagent is composed of CuSO4,
Yellow
Yellow
Yellow
Yellow
Clear
solution*
solution
solution*
solution
solution*
sodium carbonate, and sodium citrate. These compounds in the presence of heat and reducing sugars carry out the following reactions.
4 mins & 30 secs
1 min & 15 secs
4-5
10
mins*
mins*
-
Na2CO3 + 2H2O → 2NaOH + H 2CO3
Biochem 34.1 │ Isolation of Glycogen
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Theoretical positive results of carbohydrates in Osazone test. (From L to R: glucose, fructose, sucrose). (Chhabra, 2015)
Osazone test is the last qualitative test performed in the experiment. It is named as such since this detects and identifies reducing sugars like monosaccharides and disaccharides based on the formation of osazone and its formation time. Sugar osazones are yellow crystalline compounds characteristic to every reducing sugars, therefore are seen in various shapes and forms (under the microscope). Formation of these crystals under certain conditions, either hot or cold, further identifies the reducing sugar. Generally, monosaccharides give crystals on heating and all disaccharides give crystal on cooling (IMDCBiochem, 2010). In addition, the crystal formation time determines the reducing sugar in the sample. For instance, glucosazone is formed by glucose within 45 minutes of heating.
Reaction mechanism of Osazone test. (Nigam & Ayyagari, 2007)
After performing the Osazone test, the neutralized hydrolyzate was observed as a yellowcolored solution (prefer to Table 3). This is similar to the obtained result for positive controls- 1% arabinose, 1% glucose (theoretical). This confirms the presence of reducing sugar which is glucose in the sample. It can be assumed that hydrolysis of glycogen was successful and release of glucose as monomeric units was effectively executed. It is observed that the time of appearance of osazone crystals for the crude sample is within the theoretical range of the positive control (1% glucose) which verifies that glucose is present in glycogen. The time of appearance under NH is theoretically similar to the 1% glucose positive control since it is expected that the reducing sugar in the hydrolyzate is ideally glucose.
is
It is to be noted that the samples were not
phenylhydrazine reagent that is made up of phenylhydrazine and sodium acetate diluted in water.
viewed under the microscope, thus there are no are images procured in the experiment for Osazone test. Instead, theoretical image is shown below displaying
The
reagent
used
in
this
test
Sodium acetate provides a constant pH in the solution. The mechanism of this test (see Figure 7) includes the reaction of carbonyl group of the reducing carbohydrate with phenylhydrazine under
the crystal (glucosazone) formed by glucose in Osazone test.
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.
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
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mainly differ in their configuration or the functional
sugar. The latter test distinguishes reducing sugars
group at the C1 and C2 positions. Thus, the differences in these carbon positions do not affect the shapes of the crystals formed (Nigam & Ayyagari,
among each other basing on the osazone crystal formation upon reaction with phenylhydrazine 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
Reaction illustrating the formation of similar osazone from D-(+)-glucose and D-(+)-mannose. (Chhabra, 2015)
Purification and isolation of glycogen from chicken liver tissue was effectively achieved by performing homogenization, centrifugation, and precipitation (use of acetic acid and cold ethanol). Further validation was done by performing three qualitative tests. The tests include Molisch test,
Benedict’s test, and Osazone test. Molisch
test
is
a
general
It is recommended that other qualitative tests are performed to improve the analysis of the sample and its components. Additional tests may include
Barfoed’s test, Seliwanoff’s test, Bial’s-Orcinol
test, and Mucic acid test. The reagents utilized should be properly stored to avoid degradation and they should be frequently updated so that better results are achieved. Proper adherence to the given procedure is suggested to avoid any erroneous outcomes and to avoid accidents as well.
test
carbohydrates. This is based on the reaction
for of α -
naphthol compounds with furfural or its derivatives. Positive result is indicated by a purple ring at the junction of two layers in the solution. The obtained result confirms that isolated crude and hydrolyzate is a carbohydrate. However, this test is non-specific for carbohydrate because it will also give positive results for glycoproteins, glycolipids, and nucleic acids. But there is a high chance that carbohydrate is glycogen based on the certain isolation method by cold precipitation using ethanol.
Benedict’s test and Osazone test are based on the reducing properties of sugars. The former is sensitive and distinguishes between a reducing and a non-reducing sugar. Under alkaline medium and high temperature, cupric ions are reduced to cuprous ions by reducing sugars, while they in turn are oxidized to sugar acids. Formation of cuprous oxide precipitates that vary from color green, yellow, orange, red, brick-red based on the amount of
reducing sugar is the positive result for Benedict’s test. On the other hand, blue solution is a negative result. Incorrect result is obtained in the experiment since theoretically the hydrolyzate produces a positive result. Potential cause of the inaccuracy is the incomplete hydrolysis of the crude sample of glycogen which should ideally produce glucose (monomer unit of glycogen) which is a reducing
Biochem 34.1 │ Isolation of Glycogen
the theoretical range of osazone formation time for 1% glucose that boosts the confidence that the extracted sample is glycogen.
Aryal, S. (2015, October 29). Benedict's TestPrinciple, Composition, Preparation, Procedure and Result Interpretation. Interpretation. Retrieved April 2016, from Microbiology Notes: http://www.microbiologyinfo.com/benedictstest-principle-composition-preparationprocedure-and-result-interpretation/ azaquar. (2011, March 05). Carbohydrate Chemistry . Retrieved April 2016, from AzaQuar: http://www.azaquar.com/en/doc/carbohydrat e-chemistry Berg, J. M., Tymoczko, J. L., & Stryer, L. (2012). Biochemistry (Seventh ed.). New York : W. H. Freeman and Company. Campbell, M. K., & Farrell, S. O. (2015). Biochemistry (Eighth ed.). Stamford: Cengage Learning. Chhabra, D. N. (2014, June 15). Qualitative Tests for Carbohydrates - Methods and Significance. Retrieved April 2016, from Biochemistry for Medics: http://www.namrata.co/category/chemistry-ofcarbohydrates/ Chhabra, D. N. (2015, April 1). Storage polysaccharides. polysaccharides . Retrieved April 2016, from Biochemistry for Medics: http://www.namrata.co/category/chemistry-ofcarbohydrates/
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Galewski, Z., Gogiel, T., Malkowski, A., Romanowicz, L., Sobolewski, K., & Wolanska, M. (2013). Biochemistry Workbook. Bialystok. Workbook. Bialystok. Garcia, E. I., Gawaran, K. K., Herrera, M. S., Jayme, M. S., & Jimenez, R. M. (n.d.). Isolation of Glycogen and Qualitative Analysis of Glucose, Galactose, Xylose, Fructose, Lactose, Sucrose, and Starch. Glycogen. (2013, Glycogen. (2013, April 6). Retrieved April 2016, from http://csk.umed.lodz.pl/~luska/6yearprogram me/lab3a.pdf IMDCBiochem. (2010, February 10). Carbohydrates Tests Practical Handouts. Handouts. Retrieved April 2016, from Scribd.: https://www.scribd.com/doc/26652097/Carb ohydrates-Tests-Practical-Handouts Nelson, D. L., & Cox, M. M. (2013). Lehninger Principles of Biochemistry (Sixth (Sixth ed.). New York : W. H. Freeman and Company. Nigam, D. A., & Ayyagari, D. A. (2007). Lab Manual in Biochemistry, Immunology and Biotechnology. New Delhi: Tata Company Limited.
McGraw-Hill
Biochem 34.1 │ Isolation of Glycogen
Publishing
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