lab 1 130L

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Identifying the presence of macromolecules using the Benedict's test, Biuret test, and Iodine test Sukhman Sodhi 20847011 Lab Partners: Suryna Tailor & Michaela Zanette TA’s: Dan Basilla, Sarah Kowalczyk & Savera Lodhy BIOL 130L Section 036 STC 4008 Conducted on September 29, 2023, from 2:30 pm - 5:20 pm Due date: October 6, 2023

Introduction All living materials are primarily made up of carbon, hydrogen, oxygen, nitrogen, sulphur, and phosphorus. Combining these six elements can form a multitude of molecules for biological systems, which then can be further subdivided into the four major biological macromolecules: carbohydrates, lipids, proteins, and nucleic acids. The purpose of this experiment is to identify these major macromolecules in solutions using three tests, the Iodine test, the Benedict's test, and the Biuret test. The Iodine test is employed to test for starch and glycogen. This is where the original pale-yellow iodine solution turns blue-black in the presence of starch and red-brown in the presence of glycogen. The Benedict’s test is used to test for reducing sugar. If the original blue solution develops a coloured precipitate, it means a reducing sugar is present. Lastly, a Biuret test is used to indicate the presence of protein; if there’s a violet color change, amino acids are present. The negative control in all three tests is water. Materials and Methods All procedures were carried out as outlined in Identification of Macromolecules, BIOL 130L lab manual, pages 33-36 (Department of Biology, 2023). A few deviations were made to these protocols, as listed below: 1. Page 33, step 4: Instead of transfer pipetted, a new tip was used for each cup. 2. Page 34, step 1: Instead of “place 1 drop”, 50μL was transferred using a P100 micropipette. An iodine dropper was still used. 3. Page 34, step 2: 50μL was transferred using a P100 micropipette for all solutions. 4. Page 35, step 2: 1000μL (1mL) was transferred using a P1000 micropipette. 5. Page 36, step 2: 1000μL (1mL) was transferred using a P1000 micropipette. 2

Results Table 1: Observations from the Iodine test Sample Control type Observations #1: 1% glucose solution - Pale-yellow pigment #2: 0.3% glucose-1-phosphate - Pale-yellow pigment #3: 1% maltose solution - Pale-yellow pigment #4: 5% honey solution - Pale-yellow pigment #5: 1% sucrose solution - Pale-yellow pigment #6: 1% lactose solution - Pale-yellow pigment #7: 1% glycogen solution + Reddish-brown pigment #8: 1% starch solution + Black/blue pigment #9: 1% protein - Dark yellow pigment #10: beer - Dark yellow pigment #11: distilled water - Pale-yellow pigment #12: Unknown solution (#305) + Reddish-brown pigment (+) represents positive control, (-) represents negative control Table 1 highlights the colour change each sample underwent during the Iodine test. Since iodine has a pale-yellow colour, the samples with a pale-yellow pigment indicate no colour change. In the samples where there’s observed black/blue or red/brown pigment it’s evident the solution has glycogen or starch. Table 1 also indicates the positive and negative control. 3

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Table 2: Observations from Benedict’s test Sample Control type Observations #1: 1% glucose solution + Reddish-brown pigment Precipitate formed #2: 0.3% glucose-1-phosphate - Blue pigment #3: 1% maltose solution + Reddish-brown pigment Precipitate formed #4: 5% honey solution + Dark brown pigment Precipitate formed #5: 1% sucrose solution - Blue pigment #6: 1% lactose solution + Reddish-brown pigment Precipitate formed #7: 1% glycogen solution - Green pigment with yellow pigment towards the bottom #8: 1% starch solution - Turquoise-blue pigment #9: 1% protein - Blue pigment #10: beer + Orange pigment Precipitate formed #11: distilled water - Blue pigment #12: Unknown solution (#305) + Brown-copper pigment Precipitate formed (+) represents positive control, (-) represents negative control Table 2 highlights the observed colour change each sample underwent in Benedict’s test. Benedict’s solution is originally a blue solution, and if a sample continued to have a blue pigment after the test, no change was observed. This would also represent a negative control. On the other hand, if a sample develops a coloured precipitate, then the test is positive, and it indicates the presence of a reducing sugar. 4

Table 3: Observations from the Biuret Test Sample Control type Observations #1: 1% glucose solution + Light blue pigment #2: 0.3% glucose-1-phosphate - Light blue pigment #3: 1% maltose solution + Light blue pigment #4: 5% honey solution + Green pigment #5: 1% sucrose solution - Light blue pigment #6: 1% lactose solution + Light blue pigment #7: 1% glycogen solution + Light blue pigment #8: 1% starch solution - Light blue pigment #9: 1% protein - Violet pigment #10: beer + Green-yellow pigment #11: distilled water - Light blue pigment #12: Unknown solution (#305) + Light blue pigment (+) represents positive control, (-) represents negative control Table 3 highlights the observed colour changes from each sample during the Biuret Test. The original solutions used to test for protein has a light blue pigment. If there are any other colour changes it indicates the presence of a protein. The table also suggests whether each sample is a negative or positive control. 5

Discussion During this experiment, twelve samples underwent a series of tests to identify the types of macromolecules in their solutions. One of these samples was unknown, labelled as “unknown #305.” For a few of these samples, it was easy to hypothesize their outcomes. Therefore, once the test was complete and the results matched with the hypotheses it proved that the test worked effectively. Twelve solutions underwent the Iodine test where the purpose was to identify the presence of starch and glycogen. Prior to the experiment, it was evident that there would be a reddish-brown colour change in sample #7 (1% glycogen solution) and a black-blue colour change in sample #8 (1% starch solution). This proves that in this test the 1% glycogen solution and the 1% starch solution act as the positive controls. The negative control in this experiment is sample #11, distilled water since it’s widely known that H 2 O has no starch or glycogen present. Starch and glycogen are chemically identical; however, their differences lie in their molecular weight and organization. (Brust, Orzechowski, & Fettke, 2020) Starch is made of linear and coil structures made of amylose and amylopectin. On the other hand, glycogen has long glucan chains that behave similarly to amylopectin. The “ α 1,4 glucan chains are connected via α 1,6 linkages.” (Brust, Orzechowski, & Fettke, 2020). The amylose polymer has a helical structure that results in a dark blue colour. This is what leads to the reddish-brown colour. (Brust, Orzechowski, & Fettke, 2020) This hypothesis was proved when the sample was a pale-yellow pigment. Samples 1,2,3,4,5,6 and 9 also had no observable colour change. The Iodine tests work on polysaccharides with α 1,4 and α 1,6 linkages. If these linkages are not present, specifically in the samples that don’t have a colour change, it proves that starch and iodine is not present. An 6

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important observation from this test is unknown #305 acted as a positive control where there was an observed reddish-brown colour change, indicating the presence of glycogen. The twelve samples were also tested for the presence of reducing sugars using Benedict’s test. As shown in table 2 there were several samples that produced colour changes as well as coloured precipitates. Samples one, three, four, ten and twelve displace tendencies of reducing sugars, also being positive controls in this experiment. A reducing sugar is defined as a sugar that contains a ketone or aldehyde group which further allows the sugar to behave as a reducing agent. (Kunz, Lee, Schiwek, Seewald, & Methner, 2011) Sample one, 1% glucose solution, consists of an aldehyde group which is why this is a reducing sugar. Sample three, 1% maltose solution, has either an aldehyde or ketone group which can react with any hydroxyl group. (Alberts et al., 2019) Sample four, 5% honey solution, is composed of dextrose and levulose which are reducing sugars. (Heileman, 1894) Sample six, 1% lactose solution, is a disaccharide that is made up of galactose and glucose. This composition allows a proton to be donated, thus demonstrating reducing sugar characteristics. (Shaukat et al., 2010) Sample seven, 1% glycogen solution, turned to a different colour, despite it not being a reducing sugar. This could have been due to human error such as leftover residue from previous experiments. Sample ten, beer, is composed of different sugar compounds such as glucose, maltose and fructose which give beer the attributes to be a reducing sugar. (Otter & Taylor, 1967). Sample twelve is unknown solution 305, and it formed a precipitate. This means there’s a reducing sugar in this solution as well as glycogen. Most of the samples, except sample seven, have attributes that prove they’re reducing sugars. This proves that the experiment was performed effectively. The twelve samples undergo a final biuret test, where they are tested for the presence of protein. Prior to conducting this experiment, it was evident that sample nine, 1% protein, would 7

act as a positive control. As expected, sample nine turned to a violet pigment, thus proving the experiment was accurate and conducted appropriately. As shown in Table 3, there were a few others that responded to this test and acted as a positive control including samples four and ten. Sample four, 5% honey solution, has low amounts of protein, approximately 0.1 – 0.5%, nonetheless, there is still protein to react in this experiment. (Chua, Lee, & Chan, 2013) Sample ten, beer, contains proteins which is why there was an observed colour change. (Siebert & Lynn, 2005) However the colour of the beer after the test was green-yellow. Using colour theory, it can be assumed that the yellow colour of beer reacting with violet would result in a different greenish colour. The unknown sample, #305, acted as a positive control in both the Iodine test and Benedict’s test. In the Iodine test, the unknown sample behaved similarly to the 1% glycogen solution. Therefore there’s glycogen present within the sample. The unknown sample did not behave like other positive controls as it was a different colour, however, it did produce a precipitate. This proves that there are also reducing sugars within the solution. In the Biuret test, the sample acted as a negative control where there was no observed colour change. A biochemical titration of the sample can accurately quantify the solution's glycogen content. (Pelletier, Bellot, Pouysségur, & Mazure, 2013) As aforementioned sample 11, distilled water is known not to contain any macromolecules, specifically not carbohydrates, proteins, or reduced sugars. It’s expected that this would act as the negative control in all experiments. Refer to Tables 1, 2 and 3 to see the results, but this assumption was correct. This proves that all experiments were conducted appropriately, and the associated results can be trusted. 8

Overall, the “Identification of Macromolecules” consisted of testing 12 samples, where one was unknown, to test if there were any macromolecules present. The tests were run effectively however there’s always room for error. Specifically, when multiple types of tests are run, but using the same equipment, it’s easy for residue from previous tests to remain within the apparatus. This could lead to a high chance of error. This can correlate with sample seven’s results in the Benedict test, where it should not have caused a precipitate. In conclusion, this experiment required a fair understanding of core concepts regarding each test, their associated results, and what a positive and negative control was. This gave further opportunity to identify an unknown sample. 9

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References Alberts, B., Hopkin, K., Johnson, A., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2019). Essential Cell Biology . New York, ON: W.W. Norton & Company. Brust, H., Orzechowski, S., & Fettke, J. (2020). Starch and glycogen analyses: Methods and Techniques. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407607/ Chua, L. S., Lee, J. Y., & Chan, G. F. (2013). Honey protein extraction and determination by mass spectrometry. Analytical and Bioanalytical Chemistry , 405 (10), 3063–3074. doi:10.1007/s00216-012-6630-2 Heileman, W. H. (1894). A Chemical Study of Honey. A Chemical Study of Honey , 2 . Kunz, T., Lee, E., Schiwek, V., Seewald, T., & Methner, F.-J. (2011). Glucose – a Reducing Sugar? Reducing Properties of Sugars in Beverages and Food , 64 . Otter, G. E., & Taylor, L. (1967). Determination of the sugar composition of Wort and beer by gas liquid chromatography. Journal of the Institute of Brewing , 73 (6), 570–576. doi:10.1002/j.2050-0416.1967.tb03086.x Pelletier, J., Bellot, G., Pouysségur, J., & Mazure, N. M. (2013). Biochemical titration of glycogen in vitro. Journal of Visualized Experiments , (81). doi:10.3791/50465 Shaukat, A., Levitt, M. D., Taylor, B. C., MacDonald, R., Shamliyan, T. A., Kane, R. L., & Wilt, T. J. (2010). Systematic Review: Effective Management Strategies forLactose Intolerance. Annuals of Internal Medicine , 152 (12). 10

Siebert, K. J., & Lynn, P. Y. (2005). Comparison of methods for measuring protein in beer. Journal of the American Society of Brewing Chemists , 63 (4), 163–170. doi:10.1094/asbcj- 63-0163 11

lab 1 130L

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