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Recognition of Important Macromolecules

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Determination of the presence of carbohydrates and protein in aqueous solution samples

Objectives
To determine the presence of starch, glycogen, reducing sugar, peptide, and proteins by utilizing Iodine test, Benedict test, and Biuret test.

Introduction
The purpose of this experiment was to identify the presence of macromolecules by using various positive and negative controls. The principle building blocks of living organisms are essentially constructed by carbon-containing molecules in cells. (Alberts, 2009) The macromolecules including lipids, carbohydrates, proteins, and nucleic acids contribute to the most distinctive properties on living organisms. Macromolecules are known as polymers which are constructed by small organic subunits of monomers. Biological molecules get categorized into different groups that behave similarly because of the similarity of their functional groups. Macromolecules are significant to the human body as it is what we are made of.
Lipids (fatty acid molecule) are components of cell membranes and serve as a concentrated food backup in all cells. This category of biological molecules is poorly defined as it holds the common feature that the molecules are insoluble in water. This molecule is composed of an even number of carbon atoms in a carboxyl group. Droplets of triacylglycerol molecules are stored in the cytoplasm of many cells. (Alberts, 2009)

Polysaccharides is known as the high molecular weight compound form of simple sugar glucose that plants and animal store. Within plant cells, the storage consists of starch connected together by glycosidic bonds. A larger polymer in terms of molecular weight is glycogen. Through the iodine test, we are able to distinguish the presence of starch and glycogen by the use of staining. Telling from the colour of the result, a blue back is the presence of starch and a reddish brown is the presence of glycogen. (Russo, 2010)
Proteins is a molecules found in all life-forms. Proteins are built by the use of amino acids, as they folded into a unique structure and are connected head-to-tail in a long chain. (Tabarssa, 2013) A peptide bond links the amino acid from the amino group to another adjacent amino acid from the carboxyl group. In theory, the biuret test determines the presence of peptide and protein, the Cu2+ and alkali ion in the biuret test can bind to the peptide bonds present in protein to give a violet Cu2+ peptide complex.

Figure 1 the Peptide bond

Materials and Methods Results
Please refer to Biology 130L Lab Fall 2013 Manual pg. 14-18
Results
Table 1 Benedict’s test for reducing sugars
Glucose solution
Positive- Dark orange
Glucose-1-phosphate
Negative- Blue
Maltose solution
Positive- Reddish-orange
Honey solution
Positive- Brown
Sucrose solution
Negative- Blue
Lactose solution
Positive- Brownish-orange
Glycogen solution
Negative- Blue
Starch solution
Negative- Blue
Protein
Negative- Blue
Beer
Positive- Blue-> Green-> Dark yellow
Distilled water
Negative- Blue
Unknown Solution #78
Negative- Blue
Chart 1 Result of Benedict Test

Table 2 Iodine test for starch and glycogen
Glucose solution
Negative
Glucose-1-phosphate
Negative
Maltose solution
Negative
Honey solution
Negative
Sucrose solution
Negative
Lactose solution
Negative
Glycogen solution
Positive- reddish-brown (Glycogen)
Starch solution
Positive- blue-black (Starch)
Protein
Negative
Beer
Negative
Distilled water
Negative
Unknown Solution #78
Positive- reddish-brown (Glycogen)
Chart 2 Result of Iodine Test

Table 3 Biuret test for protein
Glucose solution
Negative
Glucose-1-phosphate
Negative
Maltose solution
Negative
Honey solution
Negative- orange
Sucrose solution
Negative
Lactose solution
Negative
Glycogen solution
Negative
Starch solution
Negative
Protein
Positive - purple
Beer
Negative- grey
Distilled water
Negative
Unknown Solution #78
Negative
Chart 3 Result of Biuret Test

Discussion
We performed the Benedict’s test to detect the presence of a reducing sugar by taking a sample of the 12 solutions one by one mixing with 2ml of 10% Benedict’s solution and then boiling them in a water bath. Benedict’s test identifies reducing sugars based on their ability to reduce the cupric (Cu2+) ions to cuprous oxide at basic pH. We found Solution #1, 3, 4, 6, and 10 being positive as they all experienced colour change. As we carried out the Benedict’s test, the initial blue solution develops a colour change and forms a precipitate in which ranged between the combinations of brown and red-orange.
Secondly, we performed the iodine test where we placed one drop of the 12 solutions into a spot-plate followed by a drop of iodine solution. Iodine solution is normally a very pale yellow and as starch is a coiled polymer of glucose, it turns blue-black in the presence of starch, and a

reddish-brown in the presence of glycogen. With the Iodine test, we are able to discover that the unknown tube #78 is a form of glycogen as turned reddish-brown and represented a positive control.
At last, we performed the biuret test to test for protein within the 12 solutions. There was only 1 positive solution being #9, Protein. Although there were other colour changes including solution #4 turning orange and solution #10 turning grey, these do not represent a positive control as we added 5 drops of % CuSo4 to each tube. In this test, the Cu2+ reacts with the peptide bond to produce a violet colour. The intensity of this violet colours is told to relate to the number of peptide bonds that react.

Figure 2 Structure of four amino acids.

The possible source of errors include contamination of the samples and the lack of blank samples. To improve the precision of the result, multiple measurements should be conducted. To enhance the accuracy of the outcomes, two or more different methods should be used to compare

there results. They should agree within their expected precision. Another common method to evaluate accuracy when a second method may not be readily available is spiking. Spiking is when a small but concentrated analyte is added to the unknown sample.
My results are in agreement with the literature data. The Iodine Test, Benedict Test, and Biuret Test are able to detect the presence of starch, glycogen, reducing sugar, peptide, and protein in aqueous samples by using various positive and negative controls.

Reference
Garland Science. (2009). Essential Cell Biology (3rd ed). New York and London
Mehdi Tabarsaa, Supatra Karnjanapratuma, MyoungLae Chob, Jin-Kyung Kimc, SangGuan You. (2013). Molecular characteristics and biological activities of anionic macromolecules from Codium fragile
M.V. Russo. (2010). Advance in macromolecules: perspectives and applications
(Tabarsaa, 2013)
(Russo, 2010)

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