Full Report on

Exercise 7 ISOLATION OF GLYCOGEN

And

Exercise 8 DETERMINATION OF GLYCOGEN PURITY

John Wilbern Lopez Almeria

CHEM 160.1- 4L
1st Semester, AY 2017-2018

Groupmates:

Anino, KatreenaClaire F.

Frias, Rosher

Yangco, Ellaine Jane V.

Mr. Lloyd M. Lapoot

 I. INTRODUCTION

Carbohydrate is one of the major macromolecules that are essential to living things, especially
us humans. The function of carbohydrates in our body is to provide energy, as they are the bodys
main source of fuel. All our cells and tissues in our body need carbohydrates. Once in the body,
carbohydrates are easily converted to fuel (Sheehan, n.d.).After an organism has taken in
carbohydrates in food, the carbohydrate in it is digested and broken down to its building blocks
which is glucose, and the needed glucose are then sent through the bloodstream. When the
organism has used all the needed glucose to maintain proper functioning, the remaining glucose
are excreted or stored. (Kratz, n.d) In plants the storage form of glucose is starch while in animals
the storage form of glucose is glycogen. These starch and glycogen are polysaccharides meaning
they contain about tens to hundreds of monosaccharides (Ophardt, 2003). These polysaccharides
have certain properties that are different from the properties possessed by simple sugars
(Sabularse et al., 2013).

Ethanol Precipitation is a method to isolate glycogen and precipitate out compounds such
as nucleic acids and protein from a sample such as liver or oyster. The principle behind this is
that the glycosidic bonds in glycogen are resistant to hydrolytic activity of OH-, which is produced
by the reagent KOH, at increased temperature. On the other hand, the peptide bonds in proteins,
ester bonds in lipids and phosphodiester bonds in nucleic acids would undergo hydrolysis at high
temperature and in alkaline pH (i.e. in KOH solution). The remaining glycogen solution will only
be slightly contaminated with other polysaccharides, fragments of denatured nucleic acids and
some other low molecular weight compounds. Upon addition of ethanol glycogen would
precipitate out of the solution and a relatively purified glycogen can be obtained (Glycogen,
2013).

The glycogen precipitated in the extraction is still not pure, thus has still to undergo test
for purity. For carbohydrates, one way to test for an isolated sample purity is through the Nelsons
assay and afterwards reading the absorbance at 510 nm with a spectrophotometer. The Nelsons
assay tests for the presence of reducing sugars, such as glucose, maltose and lactose. It
quantitatively determines the amount of, for example, glucose colorimetrically. Since the isolated
glycogen is not a reducing sugar, it needs to be hydrolyzed to break the glycosidic bonds and
release the glucose, which is a reducing sugar. This acid hydrolysis is done through heating the
glycogen isolate samples with alkaline copper reagent to form a rust colored precipitate. Then the
amount of cuprous oxide produced will then be determined by the addition of arsenomolybdic acid
(reduced to arsenomolybdous acid). Afterwards, an intense blue color will be observeD (Bailey,
et.al., 1992). Acid hydrolysis of glycogen, based on literatures, requires heating for 30 minutes
with the reagents HCl solution, distilled water and 1.2 N NaOH solution. Thus, if experiment set-
up has the same condition as mentioned above, the concentration of glucose can be obtained to
compute for the actual content of glucose per mass of glycogen. When this is obtained, the
percentage of the purity can now be solved since the equation for percent purity is written below.

% purity =
[(Actual mol glucos /mg glycogen) / (Theoretical mol glucose /mg glycogen)] x 100%

Thus, the aim of this exercise is to familiarize students with proper glycogen isolation and
acid hydrolysis techniques to gain knowledge in computing for the actual yield of the glycogen
and determining the percent purity of the hydrolyzed glycogen isolate.In this full report, the yield
of glycogen isolated from mussel flesh and its purity was determined and studied.
 II. METHODOLOGY

A. Isolation of Glycogen Sample Preparation.

Fresh mussels were used in the experiment. The flesh were separated from the shell and
were placed in an ice bath. After the flesh was cooled, it was homogenized. 40.30 g of the
homogenized flesh was weighed and transferred into a 125-mL Erlenmeyer flask.

Isolation of the Glycogen

Afterwards, 7.2 mL of hot 30% aqueous KOH was added to the mixture and was heated
in a boiling water bath for one hour while stirring once in a while. Distilled water (15.0 mL) was
then added to the mixture and was transferred to a 250 mL Erlenmeyer flask. In order to
precipitate the glycogen from the mixture, 30.0 mL 95% ethanol was added and swirled. It was
allowed to stand and cool to room temperature by placing it in an ice bath for 20 minutes. After
the particles had settled down, the mixture was transferred into centrifuge tubes and was
centrifuged for 5 minutes in order for the precipitate to be collected. It was important for the speed
to be half the maximum rpm and that the mass of the tubes within 0.1 g of each other. The
supernatant was discarded and the collected glycogen precipitate was dissolved in a 6.00 mL of
cold 10% TCA solution. It was then centrifuged for the complete removal of glycogen from the
tissues by removing the precipitate and retaining the supernatant. Glycogen from the supernatant
was then recovered by adding 15 mL of 95% ethanol. The mixture was left in an ice bath for 10
minutes to stand and precipitate was collected after performing centrifugation for 5 minutes. Since
the glycogen precipitate still wasnt purely white, 12 mL of 10% TCA solution was added and later
on, the mixture was centrifuged to retain the supernatant. It was then on added with 30.0 mL of
95% etOH, to recover the glycogen from the supernatant and was stood in an ice bath for 10
minutes again. After the particles settled down, centrifugation was performed in order to collect
the precipitate. The dirty white colored precipitate was dissolved I 10 mL absolute ethanol and
8mL distilled water in order to reprecipitate. It was stood in an ice bath and then the precipitate
was recovered by centrifugation. 8 mL of absolute ethanol was added to wash the collected
precipitate and it was allowed to stand in room temperature for some minutes. Glycogen
precipitate was again washed with 4.0 mL diethyl ether and was allowed to dry in a pre-weighed
watchglass. After it has dried, the weight of the isolate was determined.

B. Determination of Glycogen Purity Preparation of a Standard Curve

Glucose served as the standard for the preparation of the standard curve. It was prepared
by diluting a 20 mM glucose solution into 2.0 mM that served as the stock solution. Seven (7) test
tubes were prepared, and each was added with different concentration of glucose solution. The
amount of glucose and distilled water (in mL) used were as follows, respectively: T1 0.0, 1.0;
T2 0.1,0. 90; T3- 0.2, 0.80; T4 0.4, 0.6; T5 0.6, 0.4; T6 0.8, 0.2; T7 1.0, 0.0.

Acid hydrolysis of Glycogen

Meanwhile, hydrolysis of glycogen was performed by weighing 50 mg of glycogen isolate

in a 50 mL beaker. It was then diluted by adding 5.0 mL distilled water to have a concentration of
10 mg/mL that would serve as the stock glycogen solution. Eight test tubes were utilized with
varying concentration of the glycogen. Test tube 1 had 0.4 mL dH2O and no glycogen solution.
Test tube 2 had 0.60 distilled water with 0.40 mL glycogen solution. The rest of the test tubes (38)
had no dH2O about 0.40 mL glycogen solution was added to each. Afterwards, different amounts
of 2 N HCl solution was added to each test tubes. 0.60 mL of 2 N HCl was measured and added
to test tube 1, 0.60 mL was added to test tubes 3-8, while test tube 2 didnt contain 2 N HCl. Each
of the test tubes 1 and 2 also contained 1.00 mL of 1.2 N NaOH solution. These test tubes were
allowed to stand in room temperature while the tubes 3-8 was dispersed in a boiling water bath.
The tubes in the boiling water bath were removed at 5-minute intervals which started from 5
minutes after boiling. Then, reaction was terminated for each of the test tubes 3-8 by adding 1.0
mL of 1.2 N NaOH after the specified heating time. All the test tubes were then diluted to 8.0 mL
distilled water and were mixed using a vortex mixer. A 0.5 mL aliquot from each of the diluted
samples was obtained and diluted again to a final volume of 1.0 mL by using distilled water and
was mixed using a vortex mixer.

Nelsons Assay

Then Nelsons reagent was prepared freshly by mixing 50 mL of reagent with 2 mL reagent
B. Reagent A was prepared by weighing 12.5 g of Na2CO3, 12.5 g sodium potassium tartrate
and 100 g NA2SO4 and diluting them in a 500 mL distilled water. While the reagent b was
prepared by dissolving 7.5 g of CuSO4 in a 100 mL beaker with 50 mL distilled water and with
the addition of 1 drop of concentrated H2SO4.

Standard solution and samples were assayed simultaneously to obtain accurate results.
For all the test tubes of the standard and the sample, 1.0 mL freshly prepared Nelsons reagent
was added. The tubes were placed in a boiling water bath and were covered with marbles for 20
minutes. It was cooled to room temperature afterwards and 1.0 mL arsenomolybdate reagent was
added to each. The contents of the tubes were mixed using a vortex mixer and were allowed to
stand for 5 minutes at room temperature. Then, 7.0 mL distilled water was added to each of the
test tubes and again mixed by the vortex mixer.

Spectrophotometry

The standards and samples absorbance were read at 510 nm. The standard curve was
graphed by plotting the absorbance at 510 nm versus the glucose concentration in mol/mL.
Meanwhile, the content of the glucose in the hydrolyzed glycogen was obtained through
interpolating its absorbance values on the of the standard curve.
 III. RESULTS AND DISCUSSIONS

A. Glycogen Isolation From Mussel Flesh

Glycogen is the storage form of carbohydrates in animals. They are highly branched
polysaccharides composed of -D-glucopyranose units linked by -1,4 and -1,6 glycosidic
bonds. These bonds are resistant to hydrolytic activity of OH at elevated temperature. While as
for the peptide bonds (in proteins), ester bonds (in lipids) and phosphodiester bonds (in nucleic
acids) hydrolysis occurs at elevated temperature in alkaline pH. Thus, in order to isolate glycogen,
the unwanted biomolecules have to be removed through use of alkali solution and other
necessary reagents in high temperature (Glycogen, 2013).

Table 7.1 Stepwise observation of glycogen isolation from mussel flesh

Steps Observations

1. Separation of flesh from shell Dark brown Dark brown color with very bad odor
color
2. Homogenization of flesh mixture Viscous, turbid, light brown mixtures with
particles
3. Saponification by heating with aqueous Viscous, turbid formation of three layers
KOH (cream, yellow, cream), heterogeneous
mixture
4. Swirling during boiling water bath Viscous, turbid transition of middle layer from
yellow to dark brown, heterogeneous mixture
5. After boiling for 1 hour Opaque, turbid dark orange and brown
layers, heterogeneous mixture with
precipitate
6. Addition of water Opaque, turbid dark orange and brown
layers, heterogeneous mixture with
precipitate
7. After addition of 95% etOH Thin or smooth, dark brown homogeneous
mixture with precipitate
8. After stirring Creamy, cloudy, brown homogeneous
mixture without precipitate
9. After standing in ice bath Smooth, darker brown with creamy lower
layer, heterogeneous mixture
10. After centrifugation Heterogeneous mixture
Supernatant Smooth, brown homogeneous mixture
discarded
Precipitate Viscous, creamy, dirty white homogeneous
mixture
11. Addition of 10% TCA Viscous, creamy, dirty white homogeneous
mixture
12. After centrifugation Heterogeneous mixture
 Supernatant Opaque, formation of 4 layers (Merky brown,
dark brown, merky brown, off-white)
heterogeneous mixture
Precipitate Opaque, brown, homogeneous mixture
discarded
13. Addition of 95% etOH Opaque, brown homogeneous mixture
14. After standing in ice bath Opaque, formation of 3 layers (dark brown,
light brown, creamy brown), heterogeneous
mixture with precipitate
15. Transfer into centrifuge tubes Opaque, creamy brown homogeneous
mixture
16. After centrifugation Heterogeneous mixture
Supernatant Opaque, creamy, dirty white homogeneous
mixture discarded
Precipitate Opaque, light creamy homogeneous mixture
collected
17. After addition of 12 mL 10% TCA Light creamy homogeneous mixture
18. After centrifugation Heterogeneous mixture
Supernatant Thin, merky brown, liquid, dirty white
heterogeneous mixture collected
Precipitate Thin, solid, yellowish homogeneous
precipitate- discarded
19. After addition of 95% etOH Thick, creamy, brown, homogeneous mixture
20. Stand in ice bath Curdy heterogeneous mixture of very light
yellow and clear layers with precipitate
21. After centrifugation Heterogeneous mixture
Supernatant Thin, translucent, homogeneous mixture
discarded
Precipitate Dirty white precipitate collected
22. After dissolved in 10 mL abs etOH and Translucent white (slightly yellowish)
water heterogeneous mixture with ppt

23. Stand in ice bath
24. After centrifugation
Supernatant
Precipitate
25. Addition of 8 mL abs etOH
26. After standing in room temperature
27. After addition with diethyl ether
28. After drying in a watchglass
Clear white liquid with clear creamy
heterogeneous mixture
Heterogeneous mixture
Clear homogeneous liquid discarded
Dirty white precipitate collected
Thin, white or milky, homogenenous mixture
Clear supernatant with milky or white
precipitate, heterogeneous mixture
Clear layer, white precipitate, heterogeneous
mixture
White powder crystals

In the first part of the isolation process the mussels were homogenized using a blender
and then 30% hot aqueous KOH was added. This was in order to solubilize the tissues as it served
as a hot concentrated alkali and also to saponify fats/lipids present (Eichenberger, 2012). While
the addition of 10% TCA added was used to extract or separate glycogen from the proteins and
nucleic acids. After centrifugation, to precipitate glycogen from the mixture, 95% ethanol was
used. In the final part of the process, traces of water are needed to be removed by the addition of
absolute ethanol and diethyl ether. The difference between these two is that absolute ethanol is
more polar, making the water residues attach to these molecules in order to dehydrate the
glycogen. While diethyl ether hastens the precipitation process since it is a volatile solvent.

Table 7.2. Percent yield of glycogen from clam flesh.

Mass of watchglass 33.61 g
Mass of watchglass + glycogen 33.962 g
Mass of glycogen 0.352 g
Mass of clam flesh 40.00 g
Theoretical yield 2% (0.806 g)
Actual Yield 0.880 %
% yield 44.0%

SAMPLE CALCULATIONS

Percentage of the actual yield (g)

= mass of glycogen / mass of clam
= [(0.352g / 40.00g) x 100%] = 0.880%

Percentage of the theoritical yield (g)

= 2%

Mass of the theoritical yield (g)

= 0.02 x (40.30g) = 0.806

Percent Yield (%)

= (actual/theoretical) x 100% =( 0.880%/2% ) X 100% = 44.0%

After the drying of the precipitate, the mass of glycogen was found out to be 0.352 grams,
having an actual yield of 0.880% and a percent yield of 44.0% This would mean that 0.880% of
the mass of the mussel samples are glycogen since the actual yield is equivalent to the mass of
glycogen over the mass of fresh mussel sample. However, the percent yield measures the
efficiency of the reaction or process and since the percent yield achieved was only 44.0% this
means that the glycogen in the mussel samples werent fully recovered. This may have been due
to experimental errors brought by mismeasurement and/or human errors during handling of the
mixtures such as spillage and evaporation of liquid sample before the last step which is drying.

B. Glycogen Purity Determine

Once the glycogen was isolated, its purity will be determined by measuring it absorbance
through spectrophotometry after conducting the nelsons assay. The relevance of using nelsons
assay is that is serves as a test for presence of reducing sugars. Although glycogen is not a
reducing sugar, therefore, it needs to be hydrolyzed first to obtain its monomer units which are
glucoseglucose. To be able to dissociate the glycosidic bonds of the glycogen, the sample should
be subjected to a strong acid, which is HCl, in an elevated or high temperature (Driskell, 2012).
Thus, when glycosidic bonds are broken, the monomer units which are reducing sugars (glucose)
can now be tested with the Nelsons assay. Here, when the sugar is heated with alkaline copper
reagent, a rust colored Cu2O is formed. And its amount formed will be determined colorimetrically
by the addition of arsenomolybdic acid. Then, reduction of the arsenomolybdic acid to
arsenomolybdous acid occurs by the Cu+. An intense blue color is achieved afterwards and is
measured colorimetrically. As intensity of the color increases, the number of reducing sugars also
increases (Sabularse et al., 2013).

B.1 Preparation Of The Standard Curve

In the preparation for the standard curve, seven test tubes were filled with different
volumes of 0.0005 M glucose solution which was then diluted to 1 mL using distilled water which
resulted to different concentration of glucose per test tube. Test Tube 1 was filled only with distilled
water, tube 2 with 0.10 mL , tube 3 with 0.20 mL, tube 4 with 0.40 mL, tube 5 with 0.60 mL, tube
6 with 0.80 mL, and tube 7 with 1.00 mL 0.0005 M glucose solution. Each tubes were then
assayed with Nelsons Method and each solution was subjected to spectrophotometry to get the
absorbance at 510 nm. The data for each tubes glucose concentration and absorbance at 510
nm is shown in table 8.1 with the corresponding standard curve as shown in figure 8.1. The
calculations for glucose concentration (umol/mL) in each tube is shown below:

C1V1 = C2V2

@Test Tube 1
C2 = (C1V1 ) / (V2) =(0.5mM)(0mL)/(1.00mL) = 0 umol/mL

@Test Tube 2
C2 = (C1V1 ) / (V2) =(0.5mM)(0.10mL)/(1.00mL) = 0.05 umol/mL

@Test Tube 3
C2 = (C1V1 ) / (V2) =(0.5mM)(0.20mL)/(1.00mL) = 0.10 umol/mL

@Test Tube 4
C2 = (C1V1 ) / (V2) =(0.5mM)(0.40mL)/(1.00mL) = 0.20 umol/mL

@Test Tube 5
C2 = (C1V1 ) / (V2) =(0.5mM)(0.60mL)/(1.00mL) = 0.30 umol/mL

@Test Tube 6
C2 = (C1V1 ) / (V2) =(0.5M)(0.80mL)/(1.00mL) = 0.40 umol/mL

@Test Tube 7
C2 = (C1V1 ) / (V2) =(0.5mM)(1.00mL)/(1.00mL) = 0.50 umol/mL
Table 8.1. Absorbance of standard glucose solutions
Test Tube No. Glucose Abs510 Corrected
concentration Absorbance
(umol/mL)
1 0 0.016 0.000
2 0.05 0.047 0.031
3 0.10 0.075 0.059
4 0.20 0.132 0.116
5 0.30 0.191 0.175
6 0.40 0.282 0.266
7 0.50 0.337 0.321

STA NDA RD CURVE

0.35
y = 0.6484x - 0.0053
0.3
0.25
0.2
ABS510

0.15
0.1
0.05
0
0 0.1 0.2 0.3 0.4 0.5 0.6
-0.05
GLUCOSE CONCENTRATION (UMOL/ML)

Figure 8.1.Graph of the glucose concentration (mol/mL) over the absorbance at 510 nm.

A = -0.00529 B= 0.6484300341 r=0.9973804471

y = 0.6484300341x - 0.00529
or
Abs = 0.6484300341[glucose(umol/mL)] - 0.00529

B.2 Hydrolysis of Glycogen

As stated in the methodology, 50 mg glycogen isolate was dissolved in 5 ml distulled water

to provide the 10mg/mL glycogen solution. Eight test tubes were utilized with varying
concentration of the glycogen. Test tube 1 with 0.40 mL distilled water, 0.60 mL 2N HCl, and 1
mL 2N NaOH with no glycogen solution, which served as the control for the spectrophotometry
method, while test tube 2 has 0.60mL distilled water, 0.40 mL glycogen solution and 1 mL 2N
NaOH with no HCl. Test tubes 3 to 8 all have the same volume of glycogen solution (0.40 mL)
and HCl (0.60 mL) but only differ on the heating time. These samples were then assayed using
nelsons method and subjected to spectrophotometry to read each samples absorbance at 510
nm.
 Shown in Table 8.2 is the corrected absorbance readings of the samples obtained by
subtracting 0.09, which is the absorbance value of test tube 1 in the samples. These values were
used to determine the glucose concentration after hydrolysis of glycogen (mol/mL) of the diluted
solutions. Complete acid hydrolysis of glycogen occured after 30 minutes in boiling water bath
with HCl.

Table 8.2. Acid hydrolysis of glycogen.

Test Incubation time Abs510 Corrected Glucose Concentration
Tube No. (min) Absorbance (umol/mL)

1 ---- 0.009 0.000 --------

2 0 0.011 0.002 0.5621
3 5 0.049 0.040 3.4923
4 10 0.110 0.101 8.1959
5 15 0.142 0.133 10.6634
6 20 0.223 0.214 16.9093
7 25 0.250 0.241 18.9913
*8 30 0.293 0.284 22.3069
Calculations for glucose concentration (umol/ml)

mol/mL glucose =[(corrected absorbance - A*) / (B**)] x DF

*A =-0.00529 **B=0.6484300341
Dilution factor (DF) = (10 mL/0.4 mL) (1mL/0.5 mL) = 50
Corrected absorbance = absorbance 0.009

Test tube 1: mol/mL glucose = [(0.000 + 0.00529) / ( 0.6484300341) x 50 = 0.0000 mol/mL

Test tube 2: mol/mL glucose = [(0.002 + 0.00529) / ( 0.6484300341) x 50 = 0.5621 mol/mL
Test tube 3: mol/mL glucose = [(0.040 + 0.00529) / ( 0.6484300341) x 50 = 3.4923 mol/mL
Test tube 4: mol/mL glucose = [(0.101 + 0.00529) / ( 0.6484300341) x 50 = 8.1959 mol/mL
Test tube 5: mol/mL glucose = [(0.133 + 0.00529) / ( 0.6484300341) x 50 = 10.6634 mol/mL
Test tube 6: mol/mL glucose = [(0.214 + 0.00529) / ( 0.6484300341) x 50 = 16.9093 mol/mL
Test tube 7: mol/mL glucose = [(0.241 + 0.00529) / ( 0.6484300341) x 50 = 18.9913 mol/mL
Test tube 8: mol/mL glucose = [(0.284 + 0.00529) / ( 0.6484300341) x 50 = 22.3069 mol/mL

25
y = 0.7496x + 0.3446
20
[Glucose] umol/mL

15

10

0
0 5 10 15 20 25 30 35

Time (min)

Figure 8.2.Graph of time (min) over the glucose concentration (mol/mL).

 In figure 8.2, it can be observed that at increasing time, the amount/concentration of
glucose also increases. In the experiment, only 8 test tubes or sample were used and thus the
results were not able to show a graph that shows the levelling off which indicates that the
hydrolysis of glycogen is complete. Because of that, it was assumed that test tube 8 with heating
time of 30 min has the amount of glucose from complete hydrolysis of the glycogen sample.

Test tube 8, which was subjected to the same condition previously mentioned, became
the basis of the actual glucose content of the completely hydrolyzed sample. Its concentration
was found out to be 22.3069 mol/mL and from this, the actual mol of glucose per mg of
glycogen was computed to be 2.21531 mol/mg.

Aside from the relevant information above, the percent purity of the glycogen sample was
found to be 35.89%. This was obtained by dividing the actulal mol of glucose per mg of glycogen
by the theoretical mol of glucose per mg of glycogen as shown in the calculations below

Percent purity (%)

= (Actual mol glucose / mg of glycogen)/(Theoretical mol glucose / mg glycogen) x 100%

Theoretical mol glucose/mg glycogen

= (Total mass of glucose in glycogen (mg) x (180/162) x (1mmol/180 mg) x (1000mol/1 mmol)
= [50.0 mg x (180/162) x (1mmol/180 mg) x (1000mol/1 mmol)] / 50 mg glycogen
= 6.1728 ~ 6.17 umol/mg

Actual mol glucose/mg glycogen

=actual glucose content / glycogen stock solution
= (22.1531 umol glucose)/(10.0 mg glycogen) = 2.21531 umol/mg

Percent Purity
= [(2.21531 umol/mg) / (6.1728 umol/mg)] x 100%
= 35. 89%

The percent purity of the sample was calculated to be only 44.0 % thus, the isolated
glycogen from the mussel is not entirely pure and that contaminants such as protein and other
residues are present in the isolated sample. Also, in the isolation process the actual yield was
only 0.880 % which is lower than the expected 2 %. It just suggests that the isolation process was
not that efficient in isolating only glycogen from mussel flesh.This may be due to some
experimental errors that happen in almost all research studies including the isolation of
biomolecule that causes the impurities such as inaccurate readings of some instruments,
contaminated reagents, contaminated instruments, loss of sample content y evaporation nd
spillage which all resulted to the very low percent recovery and very low percent purity. (NOTE:
The percent recovery/actual yield of group 2 was lower with only 0.362% but was more pure
,having a percent purity of 74.86%, than the isolate that was discuss in this report) #See Last
Page for more info.

As stated, glycogen is the storage form of glucose in animals. Glucose is our primary
source of energy which we get from the breakdown of the food we eat into its components. Without
glucose, our cells will not have its fuel for work and thus, would die if glucose is not present. But
this not happen as long as we econtinue to eat food and have some extra glcose for storage as
glycogen.
 There are different types of glycogen storage diseases that affect the human bodys
glycogen metabolism and is called Glycogen storage disease (GSD). Glycogen storage disease
(GSD) type I, also known as von Gierke disease, is a group of inherited autosomal recessive
metabolic disorders of the glucose-6- phosphatase system which helps maintain glucose
homeostasis which causes build up of glucose in the body's cells. The accumulation of glycogen
in certain organs and tissues, especially the liver, kidneys, and small intestines, impairs their
ability to function normally. GSD type I may be subdivided into 2 major forms. GSD type Ia
demonstrates deficient G6Pase activity in the fresh and frozen liver tissue. GSD type Ib
demonstrates normal G6Pase activity in the frozen tissue samples and lowered activity in the
fresh specimens. Another type is GSD type III is also known as Forbes-Cori disease or limit
dextrinosis. It is an autosomal recessive disorder caused by mutations which causes deficiency
in glycogen debranchinging enzyme and limited storage of dextrin. The disease is characterized
by variable cardiac muscle, skeletal muscle and liver involvement and has different subtypes. In
contrast to GSD type I, liver and skeletal muscles are involved in GSD type III. Glycogen deposited
in these organs has an abnormal structure (Anastasopoulou, 2017).
 IV SUMMARY AND CONCLUSIONS

The isolation of glycogen involved the principle of ethanol precipitation which involved the
addition of necessary reagents which resulted to the change of color from viscous brown
heterogeneous mixture to creamy brown to dirty white to milky white then finally to a white
precipitate. The transition of these colors was due to the heat and addition of the different reagents
such as 30% aqueous KOH, 10% TCA, 95% ethanol, diethyl ether and absolute ethanol. The
principl behind this is that at a solution with KOH at high temperature causes hydrolysis of the
bonds of other large biomolecules except for glycogen and some small molecules. Upon addition
of ethanol, glycogen would precipitate out of the solution as a white precipitate.

In the experiment, after drying the white precipitate, its mass was found out to be 0.146
grams. The actual yield of isolated glycogen is 0.362%. This amount is the actual produced
product after isolation. Having a theoretical yield of 2%, its percent yield was calculated to be
18.1% which means that the experiment didnt produce as much glycogen isolate as expected
due to experimental and unavoidable errors.

Meanwhile, a standard curve was derived with the equation Abs = 0.6484300341 x
[glucose (umol/mL)] - 0.00529, having glucose as the standard, presented an increasing trend
as shown in figure 8.1. This equation was then used to determine the amount of glucose
(umol/mL) in the glycogen samples which were then subjected to spectrophotometry.The
absorbance of the hydrolyzed glycogen isolate samples subjected to increasing incubation time
from 0 to 30 with five minute intervals was found out to be also increasing. Interpolating these
absorbances to the standard curve resulted to the concentration of glucose from the hydrolyzed
glycogen isolate for the test tubes 1, 2, 3, 4, 5, 6, 7, and 8, which were 0 , 0.5621, 3.4923, 8.1959,
10.6634, 16.9093, 18.9913, and 22.3069, respectively. When plotted over the respective
absorbance, the graph was increasing. Thus, as incubation time increases, absorbance increases
too, as well as the glucose concentration obtained.

For the actual glucose content of the hydrolyzed glycogen isolate, 22.3069 mol/mL was
the concentration. This was based on the test tube 8 which was subjected to the hydrolysis
conditions of glycogen. This was the concentration of glycogen in test tube 8 since it was assumed
that test tube 8 with 30 minutes of heating time was the time where the complete hydrolysis of
glycogen started(since there were only 8 tubes).

Meanwhile, 2.21531 umol/mg was found out to be the actual content of glucose per milligram of
the glycogen isolate. Since the theoretical mol of gluose per milligram of glycogen is 6.1728
mol/mg, when the actual mol glucose per mg of glycogen is divided by this value, a purity of
35.89% is achieved. This would mean that the isolated glycogen wasnt pure enough and that it
still had contaminants and other residues possibly proteins, nucleotides or low molecular weight
oligonucleotides.

Thus, the conduction of these experiments resulted to achieving the isolate and its actual
yield, and also determining the purity of the glycogen isolate through acid hydrolysis and Nelsons
assay. Although it can be concluded that the isolation process was not 100% efficient since the
glucose from the hydrolyzed glycogen isolate isnt pure.
 V. LITERATURE CITED

Anastasopoulou, C. (2017). Glycogen Storage Diseases Types I-VII. Retrieved on October 24,
2017 at https://emedicine.medscape.com/article/1116574-overview

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