Glycogen isolation and determination of its purity

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Full Report on

Exercise 7

ISOLATION OF GLYCOGEN

and

Exercise 8

DETERMINATION OF GLYCOGEN PURITY

Angelica F. Costales

CHEM 160.1 – 2L

Midyear, 2015

Groupmates:

Alyssa Beth S. Cruz

Angel Marie H. Dargantes

Jeremy Rizza D.R. Gonzales

Laboratory Instructor:

Prof. Bong Carlo Remillion

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INTRODUCTION

In order for our organ systems to function well, energy is needed to be supplied to the body.

The energy comes in the form of carbohydrate that is an important component of the cell

membrane. Not only does it provides energy to the body, but also serve as energy storage.

Carbohydrates are organic molecules specifically carbon compounds that have aldehyde

(C-H=O) or ketone (C=O) groups. According to Stoker (2006), carbohydrates can be classified

broadly as monosaccharides and compound carbohydrates wherein the former includes simple

sugars that can’t be hydrolyzed further into smaller carbohydrates and the latter which includes

double sugars (disaccharides), few sugars (oligosaccharides), or of many sugars (polysaccharides).

Polysaccharides can either be storage or structural polysaccharides. The storage polysaccharide is

the form of carbohydrate for energy storage such as starch and glycogen. Compared to other

saccharides, these high compact polymers have a different physical and chemical properties. In

plants, polysaccharides are called starch, while in humans and animals, they are called glycogen.

Glycogens are polymers of glucose that contain glycosidic bonds. These molecules are

resistant to hydrolytic activity of OH at increased temperature. While other interactions such as

peptide bonds in proteins, ester bonds in lipids, and phosphodiester bonds in ribonucleic acids are

hydrolyzed using an alkaline pH at an elevated temperature. This way, glycogen can be isolated

and purified by addition of ethanol in a relatively high temperature.

The isolation of glycogen is necessary for conducting researches that would relate to the

availability of glycogen in the liver or other parts of the body in order to cure diseases or disorders

that would affect the glycogen metabolism and thus, dysfunction other internal organ systems such

as the heart, lungs, kidney, brain, and etc.

One way of isolating glycogen is through the use of the reagents 30% hot aqueous

potassium hydroxide (KOH), 10 % trichloroacetic acid (TCA), 95% ethanol and the washing

solvents, diethyl ether and absolute ethanol. The KOH serves as the alkaline solution that

hydrolyzes proteins, lipids and ribonucleic acids especially in high temperature. While as for the

TCA, it was mentioned in the study conducted by Roe, et.al. (1960), that alkaline residue digestion

after the four homogenizations with trichloroacetic acid solution, followed by alchoholic

precipitation, yielded an additional 45% of glycogen to the initial 57% total glycogen brought by

thorough grinding of the sample with 5% TCA. This explains the effectivity of the TCA in

extracting the glycogen from the other residues. As for the 95% ethanol, it precipitates out the

glycogen isolate that is washed by polar and non-polar solvents, absolute ethanol and diethyl ether,

respectively, to remove the traces of H2O and hasten the dehydration of the sample. Afterwards,

actual yield would be needed to calculate in order to know the amount or mass of glycogen per

mass of the sample.

Although isolation isn’t finished after precipitation. It has still has to undergo test for

purity. For carbohydrates, one way to test for an isolated samples purity is through the Nelson’s

assay and afterwards reading the absorbance at 510 nm with a spectrophotometer. The Nelson’s

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

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glycogen is not a reducing sugar, it needs to be hydrolyzed to break the glycosidic bonds and

release the glucose. 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. Another test for

reducing sugars is 3,5-dinitrosalicylic acid (DNS) which is about 10 times less sensitive than

Nelson’s assay. It is the method for determination of xylanase activity which dominates in

laboratories around the globe (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 µmol glucose / mg glycogen x 100%

Theoretical µmol glucose / mg glycogen

Thus, the aim of this exercise is to familiarize students with proper glycogen isolation and

acid hydrolysis techniques and for them to gain knowledge in computing for the actual yield of the

glycogen and determining the percent purity of the hydrolyzed glycogen isolate.

METHODOLOGY

A. Isolation of Glycogen

Sample Preparation

Fresh clams 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. 121.247 g of the

homogenized flesh was weighed and transferred into a 125-mL Erlenmeyer flask.

Isolation of the Glycogen

Afterwards, 21.60 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 wasn’t 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

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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: 1 – 0.0,1.0; 2 –

0.1,0.90; 3- 0.2, 0.80; 4 – 0.4, 0.6; 5 – 0.6, 0.4; 6 – 0.8, 0.2; 7 – 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 (3-

8) 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 didn’t 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.

Nelson’s Assay

Then Nelson’s 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 Nelson’s 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

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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 µmol/mL.

Meanwhile, the content of the glucose in the hydrolyzed glycogen was obtained through

interpolating its absorbance values on the ẋ of the standard curve.

RESULTS AND SAMPLE CALCULATIONS

Table 7.1 Stepwise observation of glycogen isolation from clam flesh.

Steps Observations

1. Separation of flesh from shell Dark brown color

2. Homogenization of flesh mixture Viscous, turbid, light brown mixtures with

particles

3. Saponification by heating with

aqueous KOH

Viscous, turbid formation of three layers

(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 ppt

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


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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


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


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


Supernatant Thin, translucent, homogeneous mixture –

discarded

Precipitate Dirty white precipitate – collected

22. After dissolved in 10 mL abs etOH

and water

Translucent white (slightly yellowish)

heterogeneous mixture with ppt

23. Stand in ice bath Clear white liquid with clear creamy

heterogeneous mixture


Supernatant Clear homogeneous liquid – discarded

Precipitate Dirty white precipitate – collected

25. Addition of 8 mL abs etOH Thin, white or milky, homogenenous mixture

26. After standing in room temperature Clear supernatant with milky or white

precipitate, heterogeneous mixture

27. After addition with diethyl ether Clear layer, white precipitate, heterogeneous

mixture

28. After drying in a watchglass White powder crystals

Table 7.2. Percent yield of glycogen from clam flesh.

Mass of watchglass 33.831 g

Mass of watchglass + glycogen 35.040 g

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Mass of glycogen 1.209 g

Mass of clam flesh 121.247 g

Actual yield 0.997 %

Theoretical yield 2 %

% Yield 49.85 %

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

Table 8.1. Absorbance of standard glucose solutions

Test tube no. Glucose concentration

(µmol/mL)

A510

1 0 0.000

2 0.2 0.172

3 0.4 0.286

4 0.8 0.558

5 1.2 0.797

6* 1.6 1.191

7* 2.0 1.180

8 - -

LR Values:

r = 0.998609822

slope =0.656077586 mL/µmol

y-intercept = 0.021439655

Note: * - not included in the graph in Figure 8.1

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Ab

sorb

ance

at

510

nm

Glucose concentration (µmol/mL)

Standard Curve of Glucose

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Figure 8.2. Graph of the glucose concentration (µmol/mL) from the hydrolyzed glycogen over

the corrected absorbance at 510 nm.

Table 8.2. Acid hydrolysis of glycogen.

Test tube no. Incubation time

min)

A510 Corrected

absorbance

Glucose

Concentration

(µmol/mL)

1 (blank) 0 0.010 0 -1.63

2 0 0.009 -0.001 1.558

3 5 0.055 0.034 0.957

4 10 0.101 0.091 5.301

5 15 0.184 0.174 11.627

6 20 0.259 0.249 17.342

7 25 0.468 0.458 33.270

8 30 0.412 0.402 29.002

SAMPLE CALCULATIONS

Calculations for actual yield

Mass in grams of the actual yield (g)

= mass of glycogen

mass of clam

= 1.209 g

121.247 g

= 0.00997 grams

Percentage of the actual yield (%)

= mass of glycogen x 100

Mass of clam

= 1.209 g x 100

121.247 g

= 0.997 %

-0.1

0

0.1

0.2

0.3

0.4

0.5

-1.63 1.558 0.957 5.301 11.627 17.342 33.27 29.002

Ab

sorb

ance

at

510

nm

Glucose concentration (µmol/mL)

Concentration of glucose in the hydrolyzed sample

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Calculation for theoretical yield

Theoretical yield of glycogen (in %) = 2%

In grams = 2% / 100

= 0.02 grams

Calculation for Percent yield

= actual yield x 100

Theoretical yield

= 0.997 % x 100

2 %

= 0.4985 x 100

= 49.85%

Linear regression values of standard curve

r = 0.998609822

slope =0.656077586 mL/µmol

y-intercept = 0.021439655

Calculation for glucose concentration

Dilution factor = (10 mL/0.4 mL) (1mL/0.5 mL)

= 50

Corrected absorbance = absorbance – 0.010

µmol/mL glucose = (corrected absorbance)ẋ * DF

For test tube 1:

µmol/mL glucose = (0.01 – 0.01)ẋ * 50

= (0)ẋ * 50

= - 1.63 µmol/mL

For test tube 2:

µmol/mL glucose = (0.009 – 0.01)ẋ * 50

= (-0.001)ẋ * 50

= 1.558 µmol/mL

For test tube 3:

µmol/mL glucose = (0.044 – 0.01)ẋ * 50

= (0.034)ẋ * 50

= 0.957 µmol/mL

For test tube 4:

µmol/mL glucose = (0.101 – 0.01)ẋ * 50

= (0.091)ẋ * 50

= 5.301 µmol/mL

For test tube 5:

µmol/mL glucose = (0.184– 0.01)ẋ * 50

= (0.174)ẋ * 50

= 11.627 µmol/mL

For test tube 6:

µmol/mL glucose = (0.259 – 0.01)ẋ * 50

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= (0.249)ẋ * 50

= 17.342 µmol/mL

For test tube 7:

µmol/mL glucose = (0.468– 0.01)ẋ * 50

= (0.458)ẋ * 50

= 33.270 µmol/mL

For test tube 8:

µmol/mL glucose = (0.412 – 0.01)ẋ * 50

= (0.402)ẋ * 50

= 29.003 µmol/mL

Calculation for actual glucose content

Actual glucose content = glucose content of test tube 8

Actual µmol/mL glucose = (0.412 – 0.01)ẋ * 50

= (0.402)ẋ * 50

= 29.003 µmol/mL

Calculation for actual µmol glucose per mg of glycogen

Actual µmol glucose per mg of glycogen

=`actual glucose content

Glycogen stock solution

= 29.003 µmol/mL

10 mg/mL

= 2.900 µmol/mg

Calculation for Percent purity

Theoretical µmol glucose / mg glycogen = (Total mass of glucose in glycogen (mg) x

180/162 x 1mmol/180 mg x 1000µmol/1 mmol)

Mass of glycogen (mg)

= 50 mg x 180/162 x 1mmol/180 mg x 1000 µmol/1 mmol

50 mg

= 6.17 µmol/mg

% purity = Actual µmol glucose / mg of glycogen x 100

Theoretical µmol glucose / mg glycogen

= 2.900 µmol/mg x 100

6.17 µmol/mg

= 47.006 %

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DISCUSSION

Glycogen is the storage form of carbohydrates in animals. They are highly branched

polysaccharides composed of α-D-glucopyranose units linked by α-1,4 and α-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.

In the first part of the isolation process, 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 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.

After the drying of the precipitate, the mass of glycogen was found out to be 1.209 grams,

having an actual yield of 0.997% and a percent yield of 49.85%. This would mean that 0.997% of

the mass of the clam samples are glycogen since the actual yield is equivalent to the mass of

glycogen over the mass of fresh clam sample. However, the percent yield measures the efficiency

of the reaction or process and since the percent yield achieved was only 49.85%, this means that

the glycogen in the clam samples weren’t fully recovered. This may have been due to experimental

errors brought by mismeasurement and/or human errors during handling of the mixtures.

Once the glycogen was isolated, its purity will be determined by measuring it absorbance

through spectrophotometry after conducting the nelson’s assay. The relevance of using nelson’s

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 glucose-

glucose. 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 Nelson’s 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.

The standard curve shown in Figure 8.2 shows an increasing trend with the glucose

concentration (µmol/mL) over the absorbance values read at 510 nm. Although three absorbance

weren’t included in the graph since its values are greater than 1.

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In Table 8.2, shown is the corrected absorbance readings of the samples obtained by

subtracting 0.010, 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 (µmol/mL) of the diluted

solutions. The corrected absorbance readings of the samples were interpolated from the standard

curve’s ẋ. Acid hydrolysis of glycogen occurs after 30 minutes in boiling water bath with HCl.

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 29.002 µmol/mL and from this, the actual µmol of glucose per mg of glycogen was computed

to be 2.90 µmol/mg.

Aside from the relevant information above, the percent purity of the glycogen sample was

found to be 47.006% pure. This was obtained by dividing the actulal µmol of glucose per mg of

glycogen by the theoretical µmol of glucose per mg of glycogen. Thus, the isolated glycogen from

the clam is not entirely pure and that contaminants such as protein and other residues are present

in the isolated sample. To correct the statement above, though the actual yield is 0.997%, we can’t

really say that this value is equivalent to the percent of glycogen in the mass of the clam samples

since the isolation process weren’t efficient and that the isolated glycogen is only 47.006% pure.

This may be due to experimental errors that happen in almost all research studies including the

isolation of biomolecule that causes the impurities.

There are different types of glycogen storage diseases that affect the human body’s

glycogen metabolism. The GSD II or also called as acid maltase deficiency or Pompe disease is

caused by an accumulation of glycogen in the lysozyme due to the deficiency of lysosomal

enzyme, α-1,4-glucosidase. It affects the human body such that progressive muscle weakness or

myopathy is felt throughout the body and thus affects various body tissues specifically the heart,

skeletal muscles, liver and nervous system. Another kind of GSD is the hepatorenal glycogenosis

(GSD Type 1). It is a genetic disease that results from a deficiency in the glucose-6-phosphatase

enzyme which causes the inability of the liver to produce free glucose from glycogen and from

gluconeogenesis. Thus, it causes sever hypoglycemia in man due to the affected two principal

metabolic mechanisms that supplies glucose to the rest of the body during fasting, creating an

increased stored glycogen in liver and kidneys. Though it can be treated by frequent or continuous

feedings of cornstarch or other carbohydrates.

SUMMARY AND CONCLUSION

The isolation of glycogen 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 30% aqueous KOH, 10% TCA, 95% ethanol, diethyl ether and absolute ethanol.

After drying the white precipitate, its mass was found out to be 1.209 grams. The actual yield of

isolated glycogen is 0.997%. This amount is the actual produced product after isolation. Having a

theoretical yield of 2%, its percent yield was calculated to be 49.85% which means that the

experiment didn’t produce as much glycogen isolate as expected due to experimental and

unavoidable errors.

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Meanwhile, the values of the standard curve, having glucose as the standard, presented an

increasing trend as shown in figure 8.1.

The absorbance of the hydrolyzed glycogen isolate subjected to increasing incubation time

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 -1.63, 1.558, 0.957, 5.301, 11.627, 17.342, 33.270, 29.002, 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, 29.003 µmol/mL was

the concentration. This was based on the test tube 8 which was subjected to the hydrolysis

conditions of glycogen. Meanwhile, 2.900 µmol was found out to be the actual content of glucose

per milligram of the glycogen isolate. Since the theoretical µmol of gluose per milligram of

glycogen is 6.17 µmol/mg, when the actual µmol glucose per mg of glycogen is divided by this

value, a purity of 47.006% is achieved. This would mean that the isolated glycogen wasn’t pure

enough. 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 Nelson’s

assay. Although it can be concluded that the isolation process wasn’t 100% efficient since the

glucose from the hydrolyzed glycogen isolate isn’t pure.

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LITERATURE CITED

Bailey, M.L., et. al. 1992. Interlaboratory testing of methods for assay of xylanase activity. Journal

of biotechnology. Pp. 257-270. Retrieved on July 14, 2015 at

http://www.sciencedirect.com/science/article/pii/016816569290074J

Driskell, J. 2012. Carbohydrates. Retrieved on July 13, 2015 at http://www.scribd.com/doc/9090

9780/Lab-Report-5-BMB#scribd

Eichenberger, E., et. al. 2012. Handbook of Experimental Pharmacology. Springer Science &

Business Media. Pp 105-106. Retrieved on July 13, 2015 at

https://books.google.com.ph/books?id=hpv6CAAAQBAJ&pg=PA104&lpg=PA104&dq

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1qP1S3KjxYAoxfI7qSYpOeZqCiA&hl=en&sa=X&ei=F1OjVdu6Ls7voASgl4DACQ&v

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Roe, J.H., et.al. 1960. Complete removal of glycogen from tissues by extraction with

trichloroacetic acid solution. The Journal of Biological Chemistry. Page 1. Retrieved on

July 14, 2015 at http://www.jbc.org/content/236/5/1244.full.pdf

Stoker, H.S., 2006. General, Organic and Biological Chemistry. Cengage Learning. Pp 551-552.

Retrieved on July 14, 2015 at

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bohydrates&f=false

Glycogen isolation and determination of its purity

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