BIOCHEMISTRY MANUAL Prepared by Admer Rey C. Dablio, v2012
Experiment 4 Score:
Isolation and General Tests of Glycogen from Chicken Liver Name: Joann H. Justiniane Groupmates: Marla C. Basa Ana Margarita L. Baytion
I
Date Performed: July 2016 Instructor’s Signature:
14,
Objectives:
II
To verify the presence of glycogen from chicken liver. To prove the existence of carbohydrate. Chemicals:
III
0.1% CH3COOH Molisch’s Reagent 0.01M Iodine
95% ethanol 12M H2SO4
Apparatus/Materials/Equipment:
Chicken liver 50 mL beaker 100 mL beaker Stirring rod 3 Test tubes Wire Gauze Scissor 25 mL Graduated cylinder
IV
Schematic Diagram of the Procedure: Please write on a short bond paper.
V
Summary of Theory
Water bath Test tube rack Test tube holder Hot plate Bunsen Burner Tong Petridish Mortar and Pestle
Z. Galewska, T. Gogiel, A. Malkowski, L. Romanowicz, K. Sobolewski, M. Wolañska cited that Glycogen is a branched homopolysaccharide composed of αD-glucose residues joined by glycosidic bonds. Straight sections contain glucose residues joined by the α-1,4 bonds, and branches are formed by α-1,6- glycosidic bonds. There is one branching for 8-10 glucose residues incorporated into the linear chain. The high degree of glycogen chain branching helps fulfil its function. It improves the solubility of the polysaccharide and increases the number of terminal glucose residues, which may be released or may serve as acceptors for the next molecules of this monosaccharide. Glycogen is the major glucose storage polymer in animals, has a highly branched structure which permits rapid release of glucose from glycogen stores. The ability to rapidly mobilize glucose is more essential to animals than to plants. Glycogen is a very compact structure that results from the curling of the polymer chains. The compactness of these polymer chains allows large amounts of carbon energy to be stored in a small volume, with little effect on cellular osmolarity. Glycogen acts as reservoir of glucose. The amount of glucose stored as glycogen in dynamic equilibrium is rapidly changing
No part of this manual may be reproduced without written permission from the Chemistry Department of the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.
BIOCHEMISTRY MANUAL Prepared by Admer Rey C. Dablio, v2012
depending on the state of the body and the organs synthesizing it which is the muscles and the liver. The carbohydrate tests used in this experiment can be divided into two classifications based on the mechanism of action which takes place on the reagents used in the tests. The first test involves the use of dehydrating acids followed by condensation of reagents. This is called the two-step analysis, which often yield highly coloured results. The second classification is making use of copper (II) ion-containing reagents. The copper (II) ions are reduced to cuprous oxide – copper (I) oxide – by the carbohydrates present in the samples. The Molisch’s Test will show positive results for all carbohydrates, with monosaccharides reacting much faster than disaccharides and polysaccharides. In this experiment, glycogen was isolated from the chicken liver by precipitation. Chicken liver is used in this experiment because it is a good source of glycogen. Glycogen can be separated from other proteins by mincing, grinding, and boiling the liver. VI
Observations Mass chicken liver = 6.0191 g
Sample Used Sample Purchased from Description of filtrate Description of precipitate
Chicken Liver Shopwise Supermarket Light yellow, stinky odor Stinky, light brown
A. Molisch’s Test Table A.1 Molisch’s Test Sample No. of drops of Molisch’s reagent 1 mL 5 Glycogen B. Iodine Test Table B.1 Iodine Test Sample No. of drops of 0.01M Iodine sol’n 1 mL 10 Glycogen VII
Observation
After adding 2 mL 12M H2SO4
Peachy solution
Hot and Purple ring formation
Observation
After Heating
Slightly Yellow
No change
Analysis According to Daniel Luzon Morris, Molisch’s Test was invented by Hans Molisch, an Australian botanist. He said that this test was base from the dehydration of carbohydrate with sulfuric acid to form furfural that will react with alpha-napthol to give a violet or purple colored solution. He further added that all carbohydrates monosaccharides, disaccharides, and polysaccharides will give a positive reaction, and nucleic acids and glycoproteins will also give a positive reaction, as all these compounds are eventually hydrolyzed to monosaccharides by strong mineral acids. Pentoses are dehydrated to furfural, while hexoxes are dehydrated to 5hydroxymethylfurfural. Either of these aldehydes, will condense with two molecules of naphthol to form a purple-colored product, as illustrated below by the example of glucose:
No part of this manual may be reproduced without written permission from the Chemistry Department of the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.
BIOCHEMISTRY MANUAL Prepared by Admer Rey C. Dablio, v2012
In Table A.1, Glycogen gave a positive result. This means that glycogen is a homopolysaccharide carbohydrate. Glycogen was dehydrated by H2SO4 to form hydroxymethylfurfural. Hydroxymethylfurfural was then condensed with two naphthol to form a purple colored product. Morris also cited that Iodine Test was used to identify glycogen and starch. Glycogen reacts with Iodine reagent to give a reddish-brown color or blue-black color. It was thought that starch and glycogen form helical coils. Iodine atoms can then fit into the helices to form a starch-iodine or glycogen-iodine complex. Starch in the form of amylose and amylopectin has less branches than glycogen. This means that the helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine complex. In Table B.1, Glycogen didn’t gave a positive result in Iodine solution. The formation of reddish-brown or blue-black color indicates the presence of glycogen. But in this experiment, the glycogen did not gave a positive result. This kind of occurrence is because of method error. The Iodine solution might have lost its function due to prolong storage in the laboratory. VIII
Conclusion Therefore, glycogen will give a positive result in molisch’s reagent and Iodine solution because it is a carbohydrate. The experiment was not successful due to method error and human error. The expected product was not formed because some of the reagent might have lost its function to react with other samples. For further experimentation of Isolation and General Tests of Glycogen from Chicken Liver, the experimenter recommend to freshly prepared all the chemicals that will be use in the experiment.
No part of this manual may be reproduced without written permission from the Chemistry Department of the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.
BIOCHEMISTRY MANUAL Prepared by Admer Rey C. Dablio, v2012
IX
References 1. Z. Galewska, T. Gogiel, A. Malkowski, L. Romanowicz, K. Sobolewski, M. Wolañska, Biochemistry Workbook for students of the Faculty of Medicine and the Faculty of Health Sciences Medical University of Bialystok, “Glycogen synthesis and degradation” pp. 95-96, 2013. 2. D. L. Morris, “Colorimetric determination of Glycogen, Disadvantages of Iodine Method” Received for publication July 15, 1998.
No part of this manual may be reproduced without written permission from the Chemistry Department of the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.
BIOCHEMISTRY MANUAL Prepared by Admer Rey C. Dablio, v2012
X
Appendix
Heating the Chicken Liver
Addition of Molisch’s reagent to a 1 mL solution
Filtration of Glycogen
Addition of Iodine solution to Glycogen
No part of this manual may be reproduced without written permission from the Chemistry Department of the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.
