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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 pigs liver was isolated and hydrolyzed by -amylase found in humans saliva. The hydrolysate was then subjected to different qualitative tests. Benedicts, Barfoeds, Seliwanoffs, and Bials-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 essential for lifes 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 -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 -amylase and subjected to the different qualitative test to determine the monosaccharide unit.

EXPERIMENTAL
Benedicts, Barfoeds, Seliwanoffs and Bials 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 30 minutes. After the tubes are cooled, the osazone crystals were then observed under a microscope.

Table 2. Test Result for Barfoeds Test

Test sample Glucose

Observations (what color of ppt) Brick red precipitate appears Brick red precipitate appears Brick red precipitate appears No precipitate forms No precipitate forms Brick red precipitate appears

Time of ppt. appearance 2 min

RESULTS AND DISCUSSIONS


Table 1. Test Results for Benedicts Test

Fructose Test sample Glucose Observations Brick red precipitate appears Brick red precipitate appears Brick red precipitate appears Brick red precipitate appears No precipitate forms Brick red precipitate appears Time of ppt. appearance 2 min Xylose

2 min

2 min

Fructose

2 min

Lactose Sucrose

--2 min

Xylose

2 min Unknown 2 min

Lactose

Key: -- indicates no precipitate is formed -2 min


Barfoeds test is a test for reducing sugars. In a 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 Barfoeds reagent is faster, forming cuprous oxide more quickly. Slightly acidic Reducing sugar + 2Cu2+ 2C u+ + oxidized sugar
Cu2O (brick red ppt)

Sucrose Unknown

Reducing sugars are usually detected by Benedicts 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.

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 produce any observations. The control also should give negative result since it does not contain carbohydrates. The experimental results match with the theory except that lactose does not give out any precipitate. In fact, Barfoeds solution contains lactic acid, thus it requires a very strong reducing agent (monosaccharide) to give positive results. Since lactose is a weaker reducing agent, it needs longer time to form the precipitate. In this case, the experimental time may not be enough or the heat supplied is not sufficient. If heating is prolonged, di(lactose) or even polysaccride may be partially hydrolyzed to produce monosaccharides to yield a positive test. The unknown precipitated. This indicates that it reducing and must not be sucrose. Table 3. Test Results for Seliwanoffs Test.

to give a red product. Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherry-red condensation product. Other sugars may produce yellow to faint pink colors. The results indicated that only fructose and sucrose contains ketone. The unknown is negative implying that it is neither of the two. Table 4. Test Result for Bials Test.

Test sample Glucose Fructose Xylose Lactose Sucrose Unknown

Observations (what color of ppt) ------Blue-green Soln. ----------

Test sample Glucose Fructose

Observations (what color of ppt) No precipitate forms Cherry red precipitate appears No precipitate forms No precipitate forms Cherry red precipitate appears No precipitate forms

Bials reagent contains orcinol (5methylresorcinol) in concentrated HCl with small amount of FeCl3. Pentoses undergo dehydration to form furfural that condenses with orcinol to form a blue-green solution. The test results indicate that only xylose is a pentose.

Xylose Lactose Sucrose

CONCLUSION
The test results indicated that the unknown solution matched the same results for glucose. It implies that the monosaccharide unit of glycogen is glucose.

Unknown

REFERENCES
Campbell, Mary; Farell Shawn. (2008). Biochemistry (6th ed.). Canada: Brooks/Crole. The Biochemistry Department (2008). Laboratory Manual in General Biochemistry. Manila: University of Santo Tomas.

Seliwanoff test can differentiate ketoses from aldoses. Ketohexose would be dehydrated by concentrated hydrochloric acid to form 5hydromethyl furfural, which then reacts with resorcinol to form a red-coloured complex. 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