Experiment 6 Biochemical Testing of Macromolecules VY NGUYEN 11 March 2016

ABSTRACT 1. Describe the structures and functions of the four main categories of biologically important macromolecules. 2. Perform chemical tests to identify the presence of lipids, proteins, two forms of carbohydrates, and DNA. 3. Understand and explain the importance of control experiments

Experiment 6: Biochemical Testing of Macromolecules Vy Nguyen 1 ​

Part I: Answer the following questions. Include questions 1­10 in your report.

1) Present a typed version of Table 1, complete with your observations. Which of the solutions is a negative control? What does this result tell you? (2 pts)

Table 1: Solutions and Color Reactions for Benedict’s Test and Iodine Test

Tube Solution Benedict’s Color Reaction Iodine Color Reaction

A 10 drops of onion juice Brown (+) Yellow­Brown (­)

B 10 drops of potato juice Yellow (+) Black (+)

C 10 drops of 1% sucrose solution Blue (­) Yellow­Brown (­)

D 10 drops of 1% glucose solution Brown­orange (+) Yellow­Brown (­) (control for Benedict’s)

E 10 drops of 1% starch solution Blue (­) Black (+) (control for Iodine)

F 10 drops of milk Yellow­orange (+) Brown (­)

G 10 drops of distilled water Blue (­) Yellow­Brown (­) (control)

The Benedict’s test is used for detecting reducing sugars, which contain aldehyde groups that are oxidized to carboxylic acids, such as glucose, fructose, glyceraldehyde, arabinose, lactose, and maltose. Ketoses can also be a reducing sugar if they contain alpha­hydroxy­ketones or if they can isomerise to aldoses during the reaction. The negative control of the Benedict’s test is distilled water, which does not contain reducing sugars (shows what a negative result looks like) ​ and appears as blue. The following solutions show the negative result of the Benedict’s test: ➢ Tube C: 10 drops of 1% sucrose solution ➢ Tube E: 10 drops of 1% starch solution (control for Iodine) ➢ Tube G: 10 drops of distilled water (control) The Iodine test is used for detecting starch. Iodine interacts with coiled polymer of glucose and becomes black. The negative control of the Iodine test is distilled water, which does not contain ​ ​ starch (shows what a negative result looks like) and appears as yellow­brown/orange. The following solutions show the negative result of the Iodine test: ➢ Tube A: 10 drops of onion juice ➢ Tube C: 10 drops of 1% sucrose solution ➢ Tube D: 10 drops of 1% glucose solution (control for Benedict’s) ➢ Tube F: 10 drops of milk ➢ Tube G: 10 drops of distilled water(control) Based on both test, there is a presence of both a reducing sugar and starch in potatoes and an absence of both a reducing sugar and starch in sucrose and distilled water. There is a presence of

Experiment 6: Biochemical Testing of Macromolecules Vy Nguyen 2 ​ a reducing sugar and an absence of starch in onion juice, glucose, and milk. In contrast, there is a presence of starch and an absence of a reducing sugar in the starch solution (which is a control for the iodine test).

2) Glucose in the urine, glycosuria, can be an indicator of diabetes. Which test could you use to determine if a patient’s urine contained glucose? (1 pt) The Benedict’s test is used for detecting reducing sugars, which contain aldehyde groups that are oxidized to carboxylic acids, such as glucose, fructose, glyceraldehyde, arabinose, lactose, and maltose. Thus, I could use the Benedict’s test to determine if a patient’s urine contains glucose.

3) How can a substance taste sweet, yet give a negative reaction with the Benedict’s test? (1pt) The Benedict’s test is only used for detecting reducing sugars, which contain aldehyde groups that are oxidized to carboxylic acids, such as glucose, fructose, glyceraldehyde, arabinose, lactose, and maltose. Sucrose is not a reducing sugar because the glycosidic bond between its components, fructose and glucose, prevents their isomerization. While sucrose tastes sweet, it is not a reducing sugar, and thus it will give a negative reaction with the Benedict’s test.

4) What have you learned about how carbohydrates are stored in onions and potatoes from the experiments performed in Table 1? (1 pt) Carbohydrates are stored as starch in potatoes and are stored as reducing sugars in onions. While our result shows that there is a presence of reducing sugar in potatoes, the yellow color illustrates that there is very little sugar in potatoes, in comparison to the brown color of onions. Thus onions primarily store its carbohydrate as reducing sugars, while potatoes primarily store its carbohydrate as starch.

5) Present a typed version of Table 2, complete with your observations. Which of the solutions in Table 2 is a positive control? What is the purpose of this control? (list 2 purposes) (2 pts)

Table 2: Solutions and Color Reactions for the Biuret Test

Tube Solution Color

H 1 ml of egg white Deep violet (+)

I 1 ml of milk Violet (+)

J 1 ml of amino acid solution (1% glycine) Blue (­)

K 1 ml of distilled water (control) Light blue (­)

L 1 ml of protein solution (1% Bovine Serum Albumin) Deep violet (+) (control)

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The protein solution (1% Bovine Serum Albumin) turns deep violet with the Biuret reagent, thus it is the positive control. The positive control shows what a positive result should look like and ensures that the experiment is done correctly (procedures, reagents, equipment).

6) Suppose you have tested an unknown sample with Biuret and Benedict’s reagents. The solution mixed with Biuret reagent is blue. The solution boiled with Benedict’s reagent is also blue. Can you conclude the identity of the sample? Why or why not? (1 pt) No, the blue solution resulted from the Biuret test means that the solution is negative (does not contain) for protein, and the blue solution resulted from the Benedict’s test means that the solution is negative (does not contain) for a reducing sugar. The solution’s identity can be sucrose, starch, or lipid.

7) What can you conclude about the molecular content of milk based on all of the tests you performed on milk? (1 pt) Based on all of the tests I performed on milk, the molecular content of milk includes a reducing sugar (positive for Benedict’s Test) and protein (positive for Biuret Test). Milk does not contain starch (negative for Iodine Test).

8) Present a typed version of Table 3, complete with your observations. What can you conclude about the function of the detergent comparing test “N” and “O” in table 3? (In other words, what did the detergent do?) (2 pts)

Table 3: Solutions and Color Reactions for the Sudan IV Test

Tube Solution Description

M 1 ml water Pale pink solution, no precipitate + 5 drops of Sudan IV

N 1 ml water Pale pink solution ( bottom layer), thin + 5 drops of Sudan IV red solution (top layer), no precipitate + 10 drops of vegetable oil

O 1 ml water Cloudy pink solution ( bottom layer), + 5 drops of Sudan IV pink/yellow precipitate layer (top + 10 drops of vegetable oil layer), presence of bubble + 5 drops of detergent (Triton X­100)

The detergent forms an emulsion, preventing water and oil to separate into two different layers.

9) Present a typed version of Table 4, complete with your observations. Could the Dische diphenylamine test tell you if a DNA sample is contaminated with RNA? Why or why not? (2 pts)

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Table 4: Dische Diphenylamine Test for DNA (DEMO ­ observed in fume hood)

Tube Solution Color

1 1 ml calf­thymus DNA solution Dark blue

3 1 ml yeast total RNA solution Light blue (precipitate)/Clear solution

4 1 ml water Clear

No, the phenylamine only reacts with the deoxyribose sugar of DNA, not the ribose sugar of RNA, thus the Dische diphenylamine test could not tell you if a DNA sample is contaminated with RNA.

10) Hand­draw (2 pt) a) The ring form of a glucose molecule b) A generic amino acid structure

c) A generic nucleotide structure d) A triglyceride molecule

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Part II: Unknown identification

1) Present the results of your tests in typed table format (with proper titles). Use the tables in this handout as examples. You should describe the color of each reaction in your tables AND indicate (+) or (­) for the test result. If you did not perform a test, indicate that in the table. (2 pts) Table 5: Identification of unknown solution

Tube Type of Test Solution Observation

X Benedict’s Test 10 drops of unknown solution Blue (­) + 0.5 ml of Benedict’s solution

X1 Benedict’s Test 10 drops of distilled water (control) Blue (­) + 0.5 ml of Benedict’s solution

Y Iodine Test 10 drops of unknown solution Brown (­) + 5 drops of Lugol’s Iodine

Y1 Iodine Test 10 drops of distilled water (control) Brown (­) + 5 drops of Lugol’s Iodine

Z Biuret Test 1 ml of unknown solution Blue (­) + 1 ml of 2.5% NaOH + 8 drops of Biuret reagent

Z1 Biuret Test 1 ml of distilled water (control) Blue (­) + 1 ml of 2.5% NaOH + 8 drops of Biuret reagent

2) Write a simple summary or conclusions paragraph (4­5 sentences) stating the identity of your unknown and how you ruled out other possibilities. (3 pts) The color of the unknown solution for the Benedict’s test, which is used to detect reducing sugars, is the same as the color for the negative control (distilled water) ­ blue; so there is an absence of reducing sugar in the unknown solution. The color of the unknown solution for the Iodine test, which is used to detect starch, is the same as the color for the negative control (distilled water) ­ brown; so there is an absence of starch in the unknown solution. The color of the unknown solution for the Biuret test, which is used to detect protein, is the same as the color for the negative control (distilled water) ­ blue; so there is an absence of protein in the unknown solution. According to the observations of the negative control (distilled water), the unknown solution also shows negative results for the Benedict’s Test, Iodine Test, and Biuret Test; Thus we can rule three of the four possibilities: glucose, starch, and BSA protein. The only remaining option, sucrose, cannot be detected using the Benedict’s Test, Iodine Test, and Biuret Test, therefore the identity of the unknown solution is sucrose. ​ ​

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REVIEW FOR FINAL EXAM 1. Know the “Polymer Principle” for carbohydrates, proteins and nucleic acids. Polymer principle ­ macromolecules are polymers consisting of many small repeating ​ molecules called monomers i. Carbohydrates ­ polymer of monosaccharides ii. Proteins ­ polymer of amino acids iii. Nucleic acids ­ polymer of nucleotides 2. Be able to recognize the structure of glucose, amino acids, and nucleotides. OK ​ 3. What are reducing sugars? And how are they identified in the laboratory? Which of the following are reducing sugars? Sugars that contain aldehyde groups that are oxidized to carboxylic acids, can be detected by Benedict’s Test: (glucose, fructose, lactose) SUCROSE IS NOT A REDUCING SUGAR. 4. What is the basis for the Diphenylamine test? Acidic conditions convert deoxyribose to molecule that binds with diphenylamine = blue i. Require boiling ii. Intensity of blue = concentration of DNA 5. Is Sudan IV fat soluble or water soluble? fat­soluble ​ Test Name Procedure Positive Negative Reactions Reactions

Benedict Copper ion complex w/ citrate in Green:+ Blue: ­ (reducing alkaline solution detects reducing Yellow: ++ sugars) sugars by changing color of benedict Orange: +++ reagent Brown: ++++

Iodine Iodine interacts w/coiled polymers of Bluish Yellow (starch) glucose (starch) Black:++ Brown/Or ange: ­

Biuret Biuret reagent (copper sulfate Slight purple: Light (proteins joined soln;NaOH) reacts + blue: ­ by peptide) w/4­6peptide(C­N) Deep violet:++

Sudan IV Sudan IV (fat­soluble dye)selectively red/emulsion pink (lipids) absorbed by lipid layer

Diphenylamine Diphenylamine reacts blue Grey (DNA) w/Deoxyribose(­H) (nucleotide’s clear pentose sugar on DNA) to produce acidic condition

6. What are negative and positive controls? And what are their functions? • Positive ­ shows that test/experiment runs correctly (variable) • Negative ­ shows what a negative result looks like (no variable)

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7. Given tubes containing tested samples, be able to classify them as positive or negative for the tests listed in the above table. OK ​