EXTRACTION AND HYDROLYSIS OF GLYCOGEN AND CHARACTERIZATION TESTS FOR CARBOHYDRATES

Gerald Calimabs, Clarence F. Carino, Nicole Kristine B. Carlos, Charis Joyce B. Cauyao, Jennifer Kierstine ChuaGroup 3 2GPharmacy Biochemistry Laboratory

ABSTRACTCarbohydrates serve as the main energy source for the human body. The objective of this experiment are as follows: to isolate polysaccharides from animal and plant sources, to perform the general tests for carbohydrates, to compare the products of acid and enzymatic hydrolyses, to illustrate the specificity of alpha-amylase on the hydrolysis of the isolated polysaccharide, to prepare the dialyzing bag used in separating the products of enzymatic hydrolysis and to examine microscopically the different osazone and mucic acid crystals. The glycogen was first extracted from chicken liver. The extract was then divided into portions, one for each of the following: acid hydrolysis, enzymatic hydrolysis, Molisch’s test and Iodine test. The qualitative test was composed of the Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s-Orcinol, Mucic Acid and Phenylhydrazone Test.

INTRODUCTIONCarbohydrates are the most abundant organic compounds in the plant world. They act as storehouses (glucose, starch, glycogen) of chemical energy. Water-soluble carbohydrates often have a sweet taste and therefore are called sugars. Another term for carbohydrate is saccharide. The objective of this experiment are as follows: to isolate polysaccharides from animal and plant sources and explain the principle involved, to perform the general tests for carbohydrates, to compare the products of acid and enzymatic hydrolyses, to illustrate the specificity of alpha-amylase on the hydrolysis of the isolated polysaccharide, to prepare the dialyzing bag used in separating the products of enzymatic hydrolysis and to examine microscopically the different osazone and mucic acid crystals.

Carbohydrates are actually polyhydroxyaldehydes and polyhydroxyketones, often but not always with the general formula (CH2O)n, where n equals 3 or more. Carbohydrates are divided into three general classes (Monosaccharide, Oligosaccharide, Polysaccharise) depending on the number of carbohydrate molecules they contain.

Monosaccharides or simple sugars are those carbohydrates, which cannot be hydrolyzed into simpler compounds. It has a general formula of CnH2nOn, with one of the carbons being the carbonyl group of either an aldehyde (aldoses) or a ketone(ketoses). Aldehydes and ketones react with alcohols to form hemiacetals. Cyclic hemiacetals form very readily when hydroxyl and carbonyl groups are part of the same molecule and that their interaction produces a ring.Glucose, fructose and xylose are common examples of monosaccharides.

A common way of representing the cyclic structure of monosaccharides is the Haworth projection, named after the English chemist Sir Walter N. Haworth. In a Haworth projection, the ring is drawn flat and viewed through its edge, with the anomeric carbon on the right and the oxygen atom to the rear.

A reducing sugar is any carbohydrate that reacts with a mild oxidizing agent under basic conditions to form an aldonic acid.(reduces the oxidizing agent). All of the examples already mentioned (glucose, fructose and xylose) are reducing sugars.

Oligosaccharides contain 2-10 monosaccharide units. Among the oligosaccharides, sucrose and lactose are of considerable biological importance.

Polysaccharides consist of large numbers of monosaccharide units bonded together by glycosidic bonds. Some examples of important polysaccharides are starch and glycogen. Glycogen acts as the energy-reserve carbohydrate for animals. It is particularly prevalent in liver and skeletal muscle. Liver glycogen is used to maintain a constant level of blood glucose.Starch is used for energy storage in plants.

Hydrated glycogen and starch are digested by the endosaccharidase alpha-amylase of saliva and pancreatic juice. Hydration of the glycogen occurs during heating and is essential for digestion.

Aside from the three general classes, carbohydrates can also be categorized as simple or complex. Simple carbohydrates are also called simple sugars and are chemically made of one or two sugars. Complex carbohydrates are also known as starches and are made of three or more linked sugars.

Carbohydrate test reagents can be divided into general classes based on the type of reaction involved. The first class is a 2-step analysis consisting of the use of dehydrating acids followed by the condensation reagents. Carbohydrates in the presence of non-oxidizing acids, undergo dehydration to form furfural or hydroxymethyl furfural. Since these aldehydes will condense with aromatic amines and phenols to give intensely colored compounds, fufural formation can be used a test for carbohydrates. This is the principle involved in Molisch’s , Bial’s-Orcinol and Seliwanoff’s Tests.

The second class of test reagents uses solutions containing copper (II) ions to detect reducing sugars. The carbohydrate reduces the copper (II) ions into copper (I) oxide. This class includes the Benedict’s and Barfoed’s test.

In the experiment, the sample solution was subjected to the general test for polysaccharides, which consists Molisch’s, and Iodine reaction test and standard carbohydrate solutions were subjected to qualitative tests which includes Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s-Orcinol, Mucic Acid and Phenlhydrazone test.

EXPERIMENTALA. Compounds tested (Samples used)Glucose is also known as D-glucose, dextrose, or grape sugar and is one of the main products of photosynthesis and starts cellular respiration.

Figure 1. Structure of Glucose

Fructose or fruit sugar is a simple ketose monosaccharide found in many foods.

Figure 2. Structure of Fructose

Xylose is a sugar isolated from wood. It is an aldopentose, which means that it contains five carbon atoms and it includes an aldehyde functional group.

Figure 3. Structure of Xylose

Lactose is the sugar found in mammalian milk. It consists of D-galactopyranose bonded by a Beta-1,4-glycosidic bond to carbon 4 of D-glucopyranose. Lactose is a reducing sugar, aldose oligosachharide.

Figure 4. Structure of Lactose

Sucrose is obtained prinicipally from the juice of sugar cane and sugar beets. In sucrose, carbon 1 of alpha-D-glucopyranose bonds to carbon 2 of D-fructofuranose by an alpha-1,2-glycosidic bond. It is a non-reducing sugar, ketose oligosaccharide.

Figure 5. Structure of Sucrose

Galactose is a sugar contained in milk. Galactose makes up half of the sugar called lactose that is found in milk.

Figure 6. Structure of Galactose

Glycogen has a branched structure with linear chains of consecutive glucose residues joined by alpha-1,4 linkages and with alpha-1,6 linkages at the branch points. The result is a fanlike structure with one terminal reducing end and many non-reducing ends.

Figure 7. Structure of Glycogen

Starch can be separated into two principal polysasccharides: amylose and amylopectin. Complete hydrolysis of both amylose and amylopectin yields only D-glucose. Amylose is composed of continuous, unbranched chains of as manyas 4000 D-glucose units joined by alpha-1,4- glycosidic bonds. Amylopectin contains chains of as many as 10,000 D-glucose units joined by alpha-1,4-glycosidic bonds.

Figure 8. Structure of Starch

Table 1.Sample Carbohydrates and their Characteristics

Glucose Simple Monosaccharide Reducing Aldose

Fructose Simple Monosaccharide Reducing Ketose

Xylose Simple Monosaccharide Reducing Aldose

Lactose Complex Oligosaccharide Reducing Aldose

Sucrose Complex Oligosaccharide Non-reducing

Ketose

Starch Complex Polysaccharide Non-reducing

Aldose

B. Procedure

1. Extraction of Glycogen:For this experiment, glycogen was extracted from chicken liver. About 3 grams of chicken liver was minced and was mixed with 12mL boiling water. The mixture was then transferred into a small beaker and was boiled for 2 minutes. This step would allow the precipitation of proteins. The mixture was grinded thoroughly until lumps were no longer visible. Then, it was heated in a boiling water bath for 30 minutes with constant addition of water to avoid drying. 1mL of 0.1% acetic acid was added to improve the precipitation of proteins. The mixture was then filtered and divided intoportions for the general test for polysaccharides, acidic and enzymatic hydrolyses. Then the glycogen precipitation by ethanol was performed. To do this, 5-10 drops of ethanol was addedto 1mL glycogen solution.

2. Hydrolysis of PolysaccharidesTo perform the acidic hydrolysis, 5 drops of concentrated HCl was mixed to 5mL of the isolate in a test tube. The opening of the test tube was covered with a marble and was kept in a water

bath for 30 minutes. The hydrolysate was then subjected to Benedict’s test.To perform the enzymatic hydrolysis, 10mL of the isolated carbohydrate was mixed with 2.3mL of saliva. It was then allowed to stand at room temperature for 30 minutes. The solution was then introduced into a dialyzing bag and it was suspended overnight in a small flask with 50mL-distilled water. After 24hours, the dialyzing bag was discarded and the solution inside the flask was concentrated using an open flame to the volume of 10mL. The presence of a reducing sugar was then tested using the Benedict’s test.

3. General Test for PolysaccharidesThere are two general tests for polysaccharides, the first one is the Molisch’s test and the other is the Iodine Reaction test. For the Molisch’s test, a few drops of Molisch’s reagent (5% alpha-naphthol in 95% ethanol) were added into 1mL glycogen solution. 2mL concentrated H2SO4 was carefully poured down the side of the tube to form a layer. The result was then noted. For the Iodine reaction test a few drops of 0.01M I2 was added into 1mL sample solution and the result was noted.

4. Qualitative test for CarbohydratesThis test used six different carbohydrate solutions, namely glucose, fructose, xylose,lactose, sucrose and starch. 5 drops of each carbohydrate solution was placed in separate test tubes and was mixed with 1mL each of the following reagents: Benedict’s, Barfoed’s, Seliwanoff’s and Bial’s-Orcinol test. The test tubes were placed in a boiling water bath and were removed after one solution already gave a positive result.

The Mucic Acid test required the mixture of 3 drops of the carbohydrate solution (galactose and lactose) and 3 drops of concentrated HNO3 on a glass slide. The specimen was heat fixed and was cooled at room temperature. The crystals were then observed under the microscope.

The Phenylhydrazone reagent was prepared by mixing 2g of phenylhydrazine hydrochloride, 3g CH3COONa, and 10mL distilled water. It was then placed in a warm water bath and was stirred until it became clear. In different test tubes, 2 drops of carbohydrate solution (glucose, fructose, xylose, lactose, sucrose and starch) were mixed with 4 drops of freshly prepared phenylhydrazine reagent. The solution was mixed well and was covered with cotton. It was then heated in a boiling water bath for 30 minutes. The tubes were cooled and the crystals were observed under the microscope.

RESULTS AND DISCUSSION:

Table 2. Results for Extraction of Glycogen and General Tests for Polysaccharides

Description Molisch’s IodineChicken Liver

Yellow clear solution

Purple ring b/w H2SO4 and glycogen

Red solution

The table above shows the results obtained for the general test for polysaccharides. The extract was obtained based from the principle that polysaccharides are considerably less soluble than sugars in aqueous alcohol; glycogen can be separated from sugars and other water-soluble compounds by the precipitation with alcohol. Purified glycogen is obtained from aqueous solution by subsequent reprecipitation with alcohol.

In the Molisch test, concentrated sulfuric acid was used as the dehydrating acid. This acid dehydrates all carbohydrates, so the test is used to distinguish between carbohydrates and non-carbohydrates. The dehydration products of carbohydrates, furfural or 5-hydroxymethylfurfural, result from the reaction of the sulfuric acid with pentoses and/or hexoses. These products condense with α-naphthol to yield a purple condensation product.

carbohydrate→dehydration product →purple product

In the Iodine reaction test starches form deeply colored blue-black complexes with iodine. Starches contain α-amylose, a helical saccharide polymer, and amylopectin. Iodine forms a large complex polysaccharide with the α-amylose helix, producing the blue-black color. Simpler oligo- saccharides and monosaccharides do not form this complex with iodine. Thus, this test can be used to distinguish starches from other carbohydrates.

The result for Molisch’s test was a purple product, which is a positive result for carbohydrates. The positive result for the Iodine Reaction test is a blue-black complexwhich is only seen in starches. Since the sample we used is not starch, the visible result should not be a blue-black complex and in the case of this particular experiment we noted a red solution for the test.

Table 3. Results for Hydrolysis and Benedict’s testResults with asterisk show false or wrong results.

Description BenedictsAcid Chicken Liver:

yellow clear solution

Green precipitate*

Enzymatic Starch: Blue Solution

Either acids or enzymes catalyze the hydrolysis of the glycosidic bonds. In the acid-catalyzed hydrolysis there is a random cleavage of bonds, with the intermediate formation of all the various possible oligosaccharides and the final conversion of these oligosaccharides to glucose.

Several enzyme catalyzed hydrolyses are more specific with respect to bonds cleaved, for example, alpha-amylase of human saliva. The alpha-amylase catalyze the rapid, random hydrolysis of internal alpha-1,4 bonds. They do not however, hydrolyze alpha-1,6 linkages, regardless of molecular size, nor do they hydrolyze maltoe. Thus glycogen is initially split by alpha-amylase action into branched dextrin’s of medium molecular weight; only small amounts if maltose is formed. The final degradation products of the action of alpha-amylase on glycogen are glucose, maltose and isomaltose. The glucose is formed by the relatively slow end cleavages of the oligosaccharides.

Benedict’s test is used as a general test for detecting reducing sugars. If the saccharide is a reducing sugar, it will reduce the copper(II) ions to copper(I) oxide, a red precipitate.

R – CHO reducing carbohydrates +2Cu2+ +5OH– →R – C O –2 carbohydrate ion +Cu2O (brick red ppt)+ 3H2O

The Benedicts test both for the acid and enzymatic hydrolysate gave a false result. Since hydrolysis should have converted the glycogen to glucose, the Benedicts test should have given a brick-red precipitate, which indicates the presence of glucose, a reducing sugar.

Table 4. Results for Qualitative Tests for CarbohydratesResults with asterisk show false or wrong results.

Sugar Benedict’s Barfoed’s Seliwanoff’s Bial’s- Orcinol

Glucose Brick red precipitate

Blue solution*

Orange solution

Moss green solution

Fructose Brick Red precipitate

Brick red precipitate

Cherry red solution

Dark brown solution

Xylose Brick Red precipitate

Brick red precipitate

Green solution

Blue green solution

Lactose Brick Red precipitate

Blue solution*

Orange solution

Brownish black solution

Sucrose Blue green solution

Blue solution

Cherry red solution

Moss green solution

Starch Blue green solution

Blue solution

Yellow solution

Brown solution

For this part of the experiment, six carbohydrate solutions were subjected to different qualitative

tests. The tests include the Benedict’s, Barfoed’s, Seliwanoff’s ,Bial’s-Orcinol, Mucic Acid, and Phenylhydrazine test.

Since the Benedict’s test was already mentioned, the discussion proceeds to Barfoed’s test. The Barfoed’s test employs the same reagent as the Benedict’s test, copper (II) oxide. The difference is that this ion is used in a slightly acidic medium.

This test can be used to distinguish between reducing monosaccharides and reducing disaccharides by observing the time that the red precipitate will form. Reducing monosaccharides cause the formation of copper (I) oxide within 2–3 minutes. Reducing disaccharides cause the formation of copper(I) oxide after approximately 10 minutes.

R – CHO reducing saccharide + 2Cu + 2H2O→R –COOH + carboxylic +Cu2O (brick red ppt )+4H

Seliwanoff’s test uses 6M hydrochloric acid as the dehydrating acid and resorcinol as the condensation reagent. Seliwanoff’s test is used to distinguish between aldoses and ketoses. When mixed with Seliwanoff’s reagent, ketopentoses and ketohexoses react within 2 minutes to form a cherry-red condensation product.

ketose→dehydration product →cherry-red product(within 2 min)

Bial’s test uses concentrated hydrochloric acid as the dehydrating acid and orcinol with a trace of iron(III) chloride as the condensation reagent. Bial’s test is used to distinguish between pentoses and hexoses. Pentoses subjected to the test yield a blue or green condensation product, while hexoses yield a muddy brown-to-gray condensation product.

pentose→dehydration product: →furfural blue or green condensation product

Aldohexoses are converted to their corresponding dicarboxylic acids in the presence of strong oxidizing agents like concentrated HNO3. The dicarboxylic acid (mucic acid) produced from the oxidation of galactose is relatively insoluble and separates out as colorless crystals. The Mucic Acid test is based on the formation of a crystalline saccharic acid that is insoluble in dilute HNO3-a reaction that is unique to galactose and galactose containing compounds.

The Phenylhydrazone test can differentiate reducing sugars by the phenylhydrazones (osazones) they form with a phenylhydrazine reagent. The osazones from different reducing sugars have characteristic crystalline forms and a definite formation time.

As discussed earlier the Benedict’s test is used to determine reducing sugars by the appearance of a brick-red precipitate. The carbohydrate solutions used all gave a correct result.

For the Barfoed’s test, the brick red precipitate was produced within 3 minutes by fructose and xylose, which indicates that they are reducing monosaccharides. Glucose as we know is also a reducing monosaccharide; it should have produced the same result as fructose and xylose. Lactose, a reducing disaccharide should have also produced a brick-red precipitate within 10 minutes time, but it also failed to do so in this experiment. The sucrose and starch solution remained blue, which was expected since they are not reducing sugars.

The test to distinguish aldoses and ketoses, Seliwanoff’s test, gave correct results. Among the samples used, only fructose and sucrose were ketoses, the rest were aldoses, so only fructose and sucrose gave a cherry-red product.

Table 5. Results for Mucic Acid TestResults with asterisk show false or wrong results.Lactose*

Galactose*

Results for this test should have shown white crystals both for lactose and galactose. Since lactose is a dimer of glucose and galactose. The nitric acid will also hydrolyze the dimer and so lactose should have also given a positive result.

Table 6. Results for Phenylhydrazone Test

Glucose

Fructose

XyloseNo picture was available

Lactose

Sucrose

Starch

REFERENCESBooks:

C.Stan Tsai, Biomicromolecules: Introduction to Structure, Function & Informatics.Hoboken, New Jersey John Wiley & Sons Inc. (2007), pp.154-165

Cantarow A., Biochemistry 3 rd Edition . W.B. Saunders Company (1962), pp. 1 -28

Clark J., Experimental Biochemistry, pp.20-27

Bettelheim, Brown, Campbell, Farrel. Introduction to General, Organic & Biochemistry. Brooks/Cole (2010), pp. 517 -539

Thomas M. Devlin. Textbook of Biochemistry with clinical correlation 6thedition.Hoboken New Jersey. Wiley-Liss (2006), pp.1056-1127

Internet:Schreck J.O., Loffredo W.M., http://www.cerlabs.com/experiments/10875404464.pdf 2-18-11

http://www.newagepublishers.com/samplechapter/000091.pdf 2-19- 11