Analysis Of Macromolecules Through Biochemical Methods

Introduction

The purpose of this lab is to detect the presence of different macromolecules in eleven known and one unknown substances, through three biochemical methods of macromolecule detection. At the end of the procedure it is possible determine the component of the unknown. These methods are the iodine test, the Benedict’s test and the Biuret test.

Macromolecules are large molecules that are created from the union of smaller molecular units, known as monomers (Campbell, 1972). These biological macromolecules are the result of the processes of living organisms.

These are usually very specific compounds that fulfill vital functions. In some cases, they work as metabolic source of energy (such as carbohydrates) and in others they are structural molecules (such as lipids) (Campbell, 1972).

The iodine test is a chemical reaction used to determine the presence or alteration of starch or glycogen. When the iodine solution reacts with starch it produces a blue-black color in starch and reddish brown with glycogen (Hansen, Wayment, Klein & Godfrey, 2017). The amylose and amylopectin are components of starch, but the amylose is linear in structure, with α (1-4) bonds, which form helices where iodine molecules meet forming a dark blue color (Miles, Morris, Orford & Ring, 1985). On the other hand, amylopectin is branched in structure, with α (1-4) (1-6) bonds, which form much shorter helices and iodine molecules are unable to get together presenting a reddish-brown color (Miles, Morris, Orford & Ring, 1985). Glycogen has a structure similar to amylopectin, but it is more branched. For that reason, the glycogen has that reddish-brown color.

Benedict's test makes it possible to determine the presence of a reducing sugar based on the reaction of a sugar with the Cu + 2 ion (Pataca, Neto, Marcucci, & Poppi, 2007). Sugars with a free carbonyl group are reducing sugars. Benedict's reagent contains sodium carbonate, copper sulfate, and sodium citrate. If a reducing sugar solution is added to the reagent and it is heated until the mixture is boiled, the sugar in alkaline solution at elevated temperatures will become in D-gluconate and its enediol (Benedict,1909). Then it breaks into two highly reducing fragments, which with their exposed electrons, will react with copper. This may take less than 5 minutes and then the change in colour is notable.

The Biuret test is based in the detection of proteins and utilizes the denominate Biuret reagent. The Biuret reagent is composed of potassium hydroxide, cupric sulfate and sodium and potassium tartrate (Lipscomb, Pinchin, Collin, Harris & Keevil, 2006). Sodium hydroxide is used to alkalize the medium, since this condition is essential for the reaction to occur (Lubran, 1978). The substance that react with proteins is cupric sulfate, while sodium tartrate has the function of not allowing the formation of copper hydroxide, which tends to precipitate and interferes with the reaction (Lipscomb, Pinchin, Collin, Harris & Keevil, 2006). If the sample contains substances that have peptide bonds (proteins), the test will be positive. A reaction is interpreted as positive when the solution turns violet. The color is produced by the formation of a complex between at least two peptide bonds that have the CO-NH group and the cupric cations (Lubran, 1978).

Materials and Methods

Materials

• Eleven known substances:
  • 1% glucose solution
  • 0.3% glucose 1 phosphate
  • 1% maltose solution
  • 5% honey solution
  • 1% sucrose solution
  • 1% lactose solution
  • 1% glycogen solution
  • 1% starch solution
  • 1% protein solution
  • Beer
  • Distilled water
• Unknown substance (#84)
• Chemical reagents:
  • Iodine solution
  • Benedict's reagent
  • Biuret reagent
  • Sodium carbonate
  • Copper sulfate
  • Sodium citrate
  • Potassium hydroxide
  • Sodium and potassium tartrate

Methods

1. Iodine Test for Starch and Glycogen:
   • Prepare each sample solution according to the specified concentration.
   • Add a few drops of iodine solution to each sample and observe any color change.
   • Record the color change and presence or absence of starch or glycogen for each sample.
2. Benedict's Test for Reducing Sugars:
   • Prepare each sample solution according to the specified concentration.
   • Add Benedict's reagent to each sample and heat until boiling.
   • Observe any color change and record the results.
3. Biuret Test for Protein:
   • Prepare each sample solution according to the specified concentration.
   • Add Biuret reagent to each sample and mix thoroughly.
   • Observe any color change, particularly a violet color, and record the results.

All experimental procedures were conducted in strict accordance with the instructions outlined in the BIOL 130L lab manual (Experiment #2: Identification of Some Macromolecules, Department of Biology, 2019). These standardized procedures were followed meticulously to ensure the accuracy and reproducibility of the results.

Results

In table 1 it can be seen the positive result of starch or glycogen in solutions number 7, 8 and 12. These solutions correspond to 1% glycogen, 1% starch and the unknown, respectively. Solutions 7 and 12 changed to a reddish-brown colour, while the number 8 changed to dark blue.

As shown in table 2, solutions 1, 3, 4, 6 and 10 contain respectively, 1% glucose, 1% maltose, 5% honey, 1% lactose and beer with positive results. In contrast, solutions remaining tested negative for Benedict´s test. The change in colour was from light blue to green, but in the honey solution it turned reddish-brown.

In table 3, it can be observed how the only solution that tested positive for the Biuret test was the 1% protein. This solution turned violet which demonstrates the presence of peptide bonds. Solution number 10, beer, showed a little change to blue yellow but still negative result. The remaining solutions present light blue color, negative result.

Discussion

In this experiment it was observed how different solutions changed their color when exposed to the different methods used: Iodine test, Benedict’s test and Biuret test. The unknown number 84 was found to contain starch, this was verified due to the color change (reddish brown) in the iodine test.

Iodine test showed the positive result of starch or glycogen in solutions that had 1% glycogen, 1% starch and the unknown. This means that solutions 7 and 12 had starch due to the change in color reddish-brown, while the solution number 8 contained glycogen because it changed to dark blue (Hansen, Wayment, Klein & Godfrey, 2017). The solution containing diluted water confirms the negative control in this procedure with a light-yellow colour. Our unknown 84 was successfully treated and it was determining that it has starch on it because of the reddish-brown pigmentation. Some common food that can contain starch or glycogen could be rice, potatoes or wheat.

Benedict’s test presented positive results in solutions 1, 3, 4, 6 and 10 containing respectively, 1% glucose, 1% maltose, 5% honey, 1% lactose and beer. This indicates, reducing sugars are present (Benedict,1909). The remain solutions tested negative for Benedict´s test. The change in colour was from light blue to green, in the honey solution it turned reddish-brown. The fact that 5% of honey turned reddish-brown but the 1% glucose turned green, should happened because of the difference in quantity. Some common food that can contain glucose are honey, sugar or fruit. Benedict’s test was used as an examination for detecting diabetes mellitus trough urine. Then, it was discovered that Benedict’s test is not a definitive test for glucose (Alexander,1969). It will give coloration even if other sugars are present in the urine, such as maltose, galactose, fructose, sucrose, etc (Alexander,1969).

Biuret test tested positive only in the 1% protein solution. The final colour was violet which verifies the presence of peptide bonds (Lubran,1978). Beer solution had a little change in color to blue yellow but still negative result. If the quantities were larger the intensity in colours would be bigger too. The remaining solutions present light blue color, negative result. Some common food that contain protein are eggs, almond or meat.

Reference List

• Alexander R. W. (1969). Diabetes mellitus--current criteria for laboratory diagnosis. California medicine, 110(2), 107–113.
• Benedict, S. R. (1909). A reagent for the detection of reducing sugars. Journal of Biological Chemistry, 5(5), 485-487.
• CAMPBELL, P. (1972). Macromolecules. Nature, 237(5355), 412-412. doi: 10.1038/237412b0
• Department of Biology. (2019). Introductory cell biology laboratory. Waterloo, Canada: University of Waterloo, 32-25.
• Hansen, C., Wayment, B., Klein, S., & Godfrey, B. (2017). Iodine–Starch test for assessment of hyperhidrosis in amputees, evaluation of different methods of application*. Disability And Rehabilitation, 40(25), 3076-3080. doi: 10.1080/09638288.2017.1367965
• Lipscomb, I., Pinchin, H., Collin, R., Harris, K., & Keevil, C. (2006). The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. Journal Of Hospital Infection, 64(3), 288-292. doi: 10.1016/j.jhin.2006.07.007
• Lubran, M. M. (1978). The measurement of total serum proteins by the Biuret method. Annals of Clinical & Laboratory Science, 8(2), 106-110.
• Miles, M. J., Morris, V. J., Orford, P. D., & Ring, S. G. (1985). The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydrate research, 135(2), 271-281.
• Pataca, L. C., Neto, W. B., Marcucci, M. C., & Poppi, R. J. (2007). Determination of apparent reducing sugars, moisture and acidity in honey by attenuated total reflectance-Fourier transform infrared spectrometry. Talanta, 71(5), 1926-1931.