Biological Macromolecules Lab Report

BIOL 130 Lab Report
Identifying Two Macromolecules - Carbohydrates and Proteins

9/26/2013
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INTRODUCTION As the name suggests, macromolecules are large molecules that make up more than 90% of the total cell mass. These biological macromolecules vary greatly in size - from several hundred to several hundred million molecular weight units - and are made up of monomer units. There are four major classes of biological macromolecules: proteins, carbohydrates, lipids, and nucleic acids (Sheeler & Bianchi, 1980). Proteins are made up of polymers of amino acids. The shape and structure of a cell is defined by proteins (Alberts et al., 1989). Carbohydrates are made up of polymers of simple sugars called monosaccharides.
Carbohydrates can also be made up of two sugars, three sugars or more, and they are called disaccharides and polysaccharides respectively (Sheeler & Bianchi, 1980). Lipids are a collection of molecules that are insoluble in water but soluble in non polar solvents. Common lipids include fatty acids, glycolipds, neutral fats, and so on (Sheeler & Bianchi, 1980). Nucleic acids store and transfer all kinds of genetic information and are polymers of nucleotides (Pollard & Earnshaw, 2004). The experiment performed was designed to identify the biological macromolecules.
These biological macromolecules are to be identified by the changes in colour through three different tests - Iodine Test for starch and glycogen, Benedict 's Test for reducing sugars, and Biuret Test for Proteins. However, only two macromolecules are being identified in this experiment - carbohydrates and proteins. There are 12 solutions to be tested in this experiment. The Iodine test is used to indentify starch and glycogen in the given solutions. Of the 12 solutions, solution 8 is a starch solution and solution 7 is a glycogen solution. Starch solutions turn blue-black when Iodine solution is added to it. This is due to the formation of polyiodide chains when the Iodine solution mixes with starch. Starch contains both amylose and amylopectin. The amylose molecules in starch form helices at the locations where the Iodine molecules assemble. This cause a dark blue-black colour change ("Starch-iodine test", 2008). Therefore, solution 8 should turn blue-black when Iodine solution is added to it since it is a starch solution. However, glycogen solutions turn red-brown when Iodine solution is added. The chemical structure of glycogen is similar to the structure of amylopectin. Glycogen is highly branched. These branches are formed through acetal linkages. It is because of the highly branched structure of glycogen that solutions of glycogen turn red-brown in Iodine solutions (Ophardt, 2003). Thus,
solution 7, a glycogen solution, should turn red-brown with the addition of Iodine solution. Solution 12 is an unknown, unknown 318. The Benedict 's Test is used to identify reducing sugars. Reducing sugars are sugars that contain free aldehyde or ketone groups that are oxidized into carboxylic acids. The Benedict 's solution contains blue Cu+2 ions. These ions react with the electrons from aldehyde or ketone group, reducing the Cu+2 ions to Cu+ ions to form a red-brown precipitate of Copper (I) Oxide (Hunt, n.d.). A change in colour to red-brown indicates the presence of sugars. Of the 12 solutions, solution 4 is a solution of honey. This solution should turn red-brown since honey contains sugars. Solutions 1(glucose solution), 3(maltose solution), 5(sucrose solution), and 6(lactose solution) should turn red-brown as well. This is because glucose, maltose, sucrose, and lactose are sugars. There should be no change in colour in the other solutions. However, since solution 12 is an unknown solution, we do not know if a colour change will take place. The Biuret Test is used to identify the presence of proteins in a solution. During the Biuret Test, 2 mL of 10% NaOH and 5 drops of 1% CuSO4 solutions are added to every test tube. Proteins are made up of polypeptides of amino acid chains linked together by peptide bonds. The polypeptide backbone includes Nitrogen from the amino group of the amino acid, the α-carbon, and the carbonyl carbon from the carboxyl group (Pollard & Earnshaw, 2004). The CuSO4 solution contains Cu+2 ions that cause protein solutions to turn violet when they stick to the Nitrogen atoms of the amino group (McRae, n.d.). Of the 12 solutions, solution 9 should turn violet because it was originally a protein solution. However, none of the other solutions should turn violet, and the colour change of the unknown 318 solution cannot be predicted yet.

MATERIALS USED
Please refer to Biology 130 Lab Manual, Department of Biology 2013, pages 13-14. No other deviations were made from this procedure.

METHODS AND PROCEDURE
Please refer to Biology 130 Lab Manual, Department of Biology 2013, pages 14-18. No other deviations were made from this procedure.

RESULTS
Table 1: Observations of the Iodine Test for Starch and Glycogen
Test Tube
Colour before
Colour after
Positive/Negative
#1 (1% glucose solution)
Clear transparent
No change(clear transparent)
Negative
#2 (0.3% glucose-1-phosphate)
Clear transparent
No change(clear transparent)
Negative
#3 (1% maltose solution)
Clear transparent
No change(clear transparent)
Negative
#4 (honey solution)
Slightly translucent
No change(clear transparent)
Negative
#5 (1% sucrose solution)
Clear transparent
No change(clear transparent)
Negative
#6 (1% lactose solution)
Clear transparent
No change(clear transparent)
Negative
#7 (1% glycogen solution)
Translucent
Red-brown
Positive

#8 (1% starch solution)
Slightly translucent
Blue-black
Positive

#9 (protein solution)
Translucent
No change(clear transparent)
Negative
#10 (beer)
Brown- yellow
No change(clear transparent)
Negative
#11 (distilled water)
Clear transparent
No change(clear transparent)
Negative
#12 (unknown solution 318)*
Slightly translucent
Red-brown
Positive

* Unknown 318 (7.5mL)

Table 2: Observations of the Benedict 's Test for Reducing Sugars
Test Tube
Colour before adding Benedict 's solution
Colour after adding Benedict 's solution
Colour change
Positive/Negative
#1 (1% glucose solution)
Clear transparent
Light Blue
Light red-brown
Positive

#2 (0.3% glucose-1-phosphate)
Clear transparent
Light blue
No change (light blue)
Negative
#3 (1% maltose solution)
Clear transparent
Light blue
Red-brown
Positive

#4 (honey solution)
Slightly translucent
Light blue
Brown
Positive

#5 (1% sucrose solution)
Clear transparent
Light blue
No change (light blue)
Negative

#6 (1% lactose solution)
Clear transparent
Light blue
Red
Positive

#7 (1% glycogen solution)
Translucent
Light blue
No change (light blue)
Negative
#8 (1% starch solution)
Slightly translucent
Light blue
No change (light blue)
Negative
#9 (protein solution)
Translucent
Light blue
No change(light blue)
Negative
#10 (beer)
Brown- yellow
Green-blue
Pale thick yellow
Positive*1

#11(distilled water)
Clear transparent
Light blue
No change
Negative

#12 (Unknown solution 318)*
Slightly translucent
Light blue
Red-orange
Positive

*1 Positive because although it did not turn red/brown, there was a colour change and a coloured precipitate formed.
* Unknown 318 (7.5mL)

Table 3: Observations for the Biuret Test for Protein
Test Tube
Colour Before
Colour After
Positive/Negative
#1 (1% glucose solution)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#2 (0.3% glucose-1-phosphate)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#3 (1% maltose solution)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#4 (honey solution)
Slightly translucent
Light yellow
Positive

#5 (1% sucrose solution)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#6 (1% lactose solution)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#7 (1% glycogen solution)
Translucent
Slightly blue(due to CuSo4)
Negative
#8 (1% starch solution)
Slightly translucent
Slightly blue(due to CuSo4)
Negative
#9 (protein solution)
Translucent
Violet
Positive

#10 (beer)
Brown- yellow
Light yellow-grey
Positive

#11 (distilled water)
Clear transparent
Slightly blue(due to CuSo4)
Negative
#12 (Unknown solution 318)*
Slightly translucent
Slightly blue(due to CuSo4)
Negative
* Unknown 318 (7.5mL)

DISCUSSION The Iodine Test was done to identify which of the 12 solutions given contained starch and glycogen. Starch is a polysaccharide manufactured by plants during photosynthesis and is comprised of glucose monomers joined in α 1, 4 linkages ("Starch", 2013). It can be separated into amylose, which comprises of about 10-20% and amylopectin which makes up the remaining 80-90% (Ophardt, 2003). Glycogen is also a polysaccharide and the primary form in which carbohydrates are stored in higher organisms like animals and human beings (Ophardt, 2003). While testing for starch and glycogen in the solutions given, Iodine was added to all drops of the 12 solutions in the spot plate. If any of the solutions contained starch, the iodine test would have changed the colour of the solution to a blue-black colour. However, if the solutions contained glycogen, the iodine test would have turned the solution into a reddish-brown. Once the Iodine was added to each solution on the spot plate, colour change in three solutions were identified. Solution 8 (1% starch solution) had turned blue-black, indicating that it contained starch. This was a positive reaction because a colour change had taken place. There was no error here because the original solution to which the Iodine solution was added was 1% starch solution. When Iodine solution is added to a starch solution, a blue-black colour is formed because the "iodine/starch complex has energy level spacings that are just so for absorbing visible light- giving the complex its intense blue color" (Senese, 2010). This dark colour is very prominent which helps detect and indicate the slightest change. Next, it was identified that solutions 7 (1% glycogen solution) and 12 (Unknown 318), where solution 12 was the unknown 318, turned reddish-brown, indicating that they contained glycogen. These were positive reactions as well due to the identifiable colour change. Solution 7 was 1% glycogen solution and thus, there was no error since it turned reddish-brown with the addition of Iodine. The highly branched structure of glycogen causes it to turn red-brown in the presence of Iodine solution (Ophardt, 2003). All other solutions showed no change in colour with the addition of Iodine solution. This tells us that out of the 12 solutions, one solution contained starch, and two other solutions contained glycogen. Next, we tested for reducing sugars with the help of Benedict 's solution.
Benedict 's solution contains Cu+2 ions that are blue in colour. This means that when Benedict 's solution is added to clear transparent or translucent solutions, the solutions turn light blue due to the presence of the Cu+2 ions. Out of the 12 solutions, solution 10 (beer) was a light brown-yellow colour, while the remaining 11 were either transparent or translucent. Thus, when Benedict 's solution was added to solution 10 (beer), it turned blue-green. It then turned into a thick pale yellow colour indicating that a reaction had taken place and it was positive. This is because although solution 10 (beer) did not turn red-brown, there was a colour change and a coloured precipitate was formed. After adding the Benedict 's solution to the remaining 11 solutions, the test tubes were put into a beaker on a hot plate and were left to boil for about 5 minutes. Five of the solutions changed from a light blue colour to different shades of red-brown. When Benedict 's solution is added to reducing sugars, the Cu+2 ions for a red-brown precipitate of Copper (I) Oxide causing all sugar solutions to turn red-brown (Hunt, n.d.). Solution 1 (1% glucose solution) turned light red-brown, solution 3 (1% maltose solution) turned red-brown, solution 4 (honey solution) turned brown almost instantly, solution 6 (1% lactose solution) turned red, and solution 12 (unknown 318) turned red orange. This indicated that
solutions 1 (1% glucose solution), 3 (1% maltose solution), 4 (honey solution), 6 (1% lactose solution), and 12 (unknown 318) consisted of reducing sugars, while solutions 2 (glucose-1-phosphate), 5(1% sucrose solution), 8(1% starch solution), 9(protein solution), and 11(distilled water) did not. Since solution 4(honey solution) turned brown almost instantly when put to boil, it must contain the highest amount of reducing sugar. There was no error in this conclusion because solution 4 was originally a solution of honey. However, solution 5(1% sucrose solution) did not have a colour change although it was a solution of sucrose, a sugar. Lastly, we tested for proteins using the Biuret Test. 2mL of 10% NaOH solution was added to each of the 12 solutions in different test tubes. Next, 5 drops of 1% CuSO4 were added into the same 12 test tubes. The presence of proteins in any of the 12 solutions would have caused the solutions to turn violet after the addition of the NaOH and CuSO4 solutions because the Cu+2 ions stick to the Nitrogen molecule of the amino acid (McRae, n.d.). Solution 9(protein solution) turned purple indicating the presence of protein molecules. This was a positive reaction and there was no error because solution 9 was originally a protein solution. However, solution 4(honey solution) turned into a light yellow colour, and solution 10 (beer) turned into a light yellow-grey colour. All other solutions had no colour change indicating that proteins were not present. All the three tests show that unknown 318 is a carbohydrate. This is because while testing for starch and glycogen, unknown 318 showed the presence of glycogen, a carbohydrate. Moreover, unknown 318 also tested positive for presence of reducing sugars during the Benedict 's Test. Sugars are also carbohydrates. Nonetheless, unknown 318 tested negative for the presence of proteins which is a different macromolecule. Therefore, in conclusion, unknown 318 is a carbohydrate.

REFERENCES

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Department of Biology. (2013, Fall). BIOLOGY 130L Cell Biology Laboratory Laboratory Manual Waterloo: University of Waterloo.
Hunt, Dr. I. (n.d.). Ch25: Reducing sugars. Home | Chemistry | University of Calgary. Retrieved September 25, 2013, from http://www.chem.ucalgary.ca/courses/350/Carey5th/Ch25/ch25-2-5.html
McRae, M. (n.d.). SBE Activity. Welcome to the Commonwealth Scientific and Industrial Research Organisation (CSIRO) | CSIRO. Retrieved September 25, 2013, from http://www.csiro.au/helix/sciencemail/activities/ProteinSolution.html
Ophardt, C. (2003). Carbohydrates - Glycogen. (2003).Elmhurst College: Elmhurst, Illinois. Retrieved September 24, 2013, from http://www.elmhurst.edu/~chm/vchembook/5 47glycogen.html
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