ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES

(ENZYME HYDOLYSIS OF GLYCOGEN)

Group # 7, 2BMT, Mary Grace Saba, *Miguel M. Sabillena, Regine San Jose, David Santos, Virli-Anne
Sebastian

ABSTRACT
Glycogen form a pig’s liver was isolated and hydrolyzed by α-amylase found in
human’s saliva. The hydrolysate was then subjected to different qualitative tests. Benedict’s,
Barfoed’s, Seliwanoff’s, and Bial’s-Orcinol tests were done with different saccharide
standards (glucose, fructose, xylose, starch, sucrose, lactose) along with the unknown
hydrolysate to determine the monomer unit of glycogen.

INTRODUCTION

Carbohydrates are one of four macromolecules EXPERIMENTAL

essential for life’s processes. It provides energy
to comply with different biological processes. It
is composed of the formula (CH2O) n. It is
classified as aldoses or ketoses depending on
the presence of the functional group either on
the C1 or C2 carbon. The general classes of
carbohydrates based on the number of
carbohydrate molecule they contain are
monosaccharide, oligosaccharides, and
polysaccharides. Monosaccharide is usually the
monomers of carbohydrate because it only
contains one sugar unit which cannot be
hydrolyze. Examples of monosaccharide are
glucose, fructose, and galactose.
Oligosaccharides are composed of two to ten
monosaccharide units. Sucrose, lactose, and
maltose are classified as oligosaccharides.
Polysaccharides contain more than ten
monosaccharide units. Glycogen and starch are
examples of polysaccharides.
Polysaccharides and oligosaccharides can be
further hydrolyzed to form monosaccharide
unit. The enzyme α-amylase can be used to
hydrolyze the polysaccharide. This enzyme classified
as an endoglycosidase, hydrolyze a glycosidic linkage
anywhere along the chain to produce the
monosaccharide unit of the polysaccharide.
Glycogen is a polysaccharide found in animals
usually in the liver and skeletal muscle. It is a
branched-chain polymer composed of many but one
type of monosaccharide unit which similar to the
amylopectin fraction of starch. This polysaccharide is
hydrolyzed by the α-amylase and subjected to the
different qualitative test to determine the
monosaccharide unit.
Benedict’s, Barfoed’s, Seliwanoff’s and Bial’s
Orcinol Tests
In separate test tubes, five drops of the
carbohydrate solution (glucose, fructose,
xylose, lactose, sucrose, starch) and 1 mL of
the reagent required were mixed for each test.
Each test was done at the same time for the
carbohydrate solutions. All the test tube for a
test was placed in a boiling water bath. The test
tubes were removed when a solution for one
test give visible result. The visible result and
the time it takes to have a positive result were
taken note of.
Mucic Acid Test
Three drops of carbohydrate solution
(galactose, lactose) and three drops of
concentrated HNO3 were mixed on a glass slide.
It was passed over a small flame until almost
dry and cooled at room temperature. The
crystals formed as seen in the microscope were
examined and drew.
Phenylhydrazone Test
A phenylhydrazone reagent was prepared by
mixing 2 g phenyl hydrazine hydrochloride, 3 g
CH3COONa, and 10 mL distilled water. It was
then placed in a warm water bath until the
solution cleared. In different test tubes
containing 2 drops carbohydrate solution
(glucose, fructose, xylose, lactose, sucrose, and
starch) 4 drops of the freshly prepared reagent
was added. It was mixed well and stopper with
cotton. It was heated in a boiling water bath for Table 2. Test Result for Barfoed’s Test
30 minutes. After the tubes are cooled, the Test Observations Time of ppt.
osazone crystals were then observed under a sample (what color of ppt) appearance
microscope.
Glucose Brick red 2 min
RESULTS AND DISCUSSIONS precipitate
appears
Table 1. Test Results for Benedict’s Test
Fructose Brick red 2 min
precipitate
Test Observations Time of ppt. appears
sample appearance
Xylose Brick red 2 min
Glucose Brick red 2 min precipitate
precipitate appears
appears
Lactose No precipitate --
Fructose Brick red 2 min forms
precipitate
Sucrose No precipitate --
appears
forms
Xylose Brick red 2 min
Unknown Brick red 2 min
precipitate
precipitate
appears
appears
Lactose Brick red 2 min
Key: ‘--’ indicates no precipitate is formed
precipitate
appears

Sucrose No precipitate -- Barfoed’s test is a test for reducing sugars. In a

forms
Unknown Brick red 2 min
precipitate
appears
Reducing sugars are usually detected by
Benedict’s reagent, which contains copper (II)
ions in alkaline solution with sodium citrate
added to keep the cupric ions in the solution.
The alkaline conditions of this test causes
isomeric transformation ketoses to aldoses,
resulting in all monosaccharides and most
disaccharides reducing the blue cupric ion to
cuprous oxide (Cu2O), a brick red precipitate.
slightly acidic condition, reducing sugars would
reduce Cu(II) ions in the Barfoed’ reagent to Cu(I)
ions when heated, which will then be precipitated out
as brick red precipitat, Cu2O(s). It is also used to
distinguish between mono- and oligosaccharides and
polysacchrides by their precipitate formation rate.
Since monosacchride has higher reducing power, so
its reaction with cupric acetate in Barfoed’s reagent is
faster, forming cuprous oxide more quickly.
Slightly acidic
2C u+ + oxidized sugar
Reducing sugar + 2Cu2+
Cu2O
(brick red ppt)
All monosaccharides and reducing sugars,
including glucose, fructose, galactose and lactose
should give a positive result, because they all contain
either reducing ends or potential free aldehyde group
for reduction of Cu(II) ions . Sucrose should give a
negative result because all anomeric carbons of both
its glucose and fructose subunits are involved in the
formation of glycosidic linkage. There is no free
anomeric carbon presented for reduction. And
polysaccharides would have the lowest reducing
power because there are only very few reducing ends
in their long chain structure, so it is not enough to sucrose) form a cherry-red condensation product.
produce any observations. The control also should Other sugars may produce yellow to faint pink colors.
give negative result since it does not contain The results indicated that only fructose and
carbohydrates. sucrose contains ketone. The unknown is negative
implying that it is neither of the two.
The experimental results match with the theory
except that lactose does not give out any precipitate.
In fact, Barfoed’s solution contains lactic acid, thus it Table 4. Test Result for Bials Test.
requires a very strong reducing agent Test Observations
(monosaccharide) to give positive results. Since sample (what color of ppt)
lactose is a weaker reducing agent, it needs longer
time to form the precipitate. In this case, the Glucose ----
experimental time may not be enough or the heat
supplied is not sufficient. If heating is prolonged, di- Fructose ----
(lactose) or even polysaccride may be partially
hydrolyzed to produce monosaccharides to yield a
Xylose Blue-green Soln.
positive test.

The unknown precipitated. This indicates that it

Lactose ----
reducing and must not be sucrose.
Sucrose ----
Table 3. Test Results for Seliwanoff’s Test.
Unknown ----
Test Observations
sample (what color of ppt)
Bial’s reagent contains orcinol (5-
Glucose No precipitate methylresorcinol) in concentrated HCl with
small amount of FeCl3. Pentoses undergo
forms
dehydration to form furfural that condenses
with orcinol to form a blue-green solution.
Fructose Cherry red
The test results indicate that only xylose is a
precipitate pentose.
appears
CONCLUSION
Xylose No precipitate
forms
The test results indicated that the unknown
Lactose No precipitate solution matched the same results for glucose.
forms It implies that the monosaccharide unit of
glycogen is glucose.
Sucrose Cherry red
precipitate
appears REFERENCES
Unknown No precipitate
forms Campbell, Mary; Farell Shawn. (2008).
Biochemistry (6th ed.). Canada:
Brooks/Crole.
Seliwanoff test can differentiate ketoses from
aldoses. Ketohexose would be dehydrated by The Biochemistry Department (2008).
concentrated hydrochloric acid to form 5- Laboratory Manual in General
hydromethyl furfural, which then reacts with Biochemistry. Manila: University of
resorcinol to form a red-coloured complex. Santo Tomas.
Seliwanoff's reagent contains resorcinol in 6 M
hydrochloric acid. Hexoses undergo dehydration
when heated in this reagent to form
hydroxymethylfurfural that condenses with resorcinol
to give a red product. Ketohexoses (such as fructose)
and disaccharides containing a ketohexose (such as