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Pre-Lab 6: Biochemical Testing of Macromolecules (10 pts) Name: _______________________________ Lab Section: _______________ Grade: _______ 1. Which a. b. c. of the following is a mismatch between the subunit and the polymer? Amino acids ------- protein Deoxyribonucleotides ------ RNA Glucose ------- starch or glycogen 2. Which a. b. c. d. test will indicate the presence of starch? Biuret test Benedict’s test Iodine test None of the above 3. To test for the presence of proteins, you use _______________________. 4. Benedict’s solution is commonly used to test for the presence of _________________. 5. In Biuret’s test, what indicates a positive result? 6. Which a. b. c. d. of the following is NOT a reducing sugar? Fructose Glucose Lactose Sucrose 7. Sudan a. b. c. d. IV stains the lipid layer what color? Blue Red Purple Black 8. Can the diphenylamine test differentiate DNA and RNA? Why or why not? 9. Name the two tests in this experiment that require boiling. 10. Explain the difference between a negative control and a positive control. BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report Experiment 6: Biochemical Testing of Macromolecules Objectives: At the end of this exercise, you should be able to 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. Introduction Cells are composed of five broad categories of chemical molecules: inorganic molecules, carbohydrates, lipids, proteins and nucleic acids. The last 4 are organic molecules. For the most part, each of these four types of macromolecules is composed of smaller subunits held together by covalent bonds resulting in very large molecules (macromolecules). These macromolecules then provide much of the structure and function of living cells. Carbohydrates: Carbohydrates are composed primarily of the elements carbon (C), hydrogen (H) and oxygen (O) at a ratio of 1:2:1, respectively. An important characteristic of carbohydrates is that they are polyhydroxyl aldehydes and ketones, meaning that they have a double-bonded oxygen (=O) attached to one of their carbon atoms and hydroxyl (-OH) groups attached to the others. “Simple sugars” is just another name for mono- and disaccharides: sweet substances with which we are familiar, such as glucose, fructose, and sucrose (table sugar). Polymers of simple sugars bound by covalent bonds are called polysaccharides. Starch, glycogen, and cellulose are all examples of polysaccharides. Carbohydrates function primarily as energy storage and structural molecules in cells. Benedict’s reagent can be used for a certain class of sugars, the reducing sugars. Reducing sugars possess free aldehyde (-CHO) or ketone (-C=O) groups that reduce weak oxidizing agents such as Benedict’s reagent. Most monosaccharides, but only some disaccharides, are reducing sugars. Benedict’s reagent contains a cupric (copper) ion complexed with citrate in an alkaline solution. It will react with reducing sugars, turning the solution from blue to green in the presence of small amounts of these molecules, and turning the solution yellow-orange or even red in the presence of larger amounts. The iodine test is used to determine the presence of starch. The basis for this test is that starch is a coiled polymer of glucose; iodine (iodine-potassium iodide, I2KI) interacts with these coiled molecules and becomes bluish-black. A yellowish-brown color (i.e. no color change) is a negative test for starch. Proteins: Proteins are polymers of amino acids joined together by peptide bonds. Each amino acid contains a carbon to which an amino group (-NH2), a hydrogen (H), a carboxyl group (-COOH) and another variable group (generically designated as –R), that varies in each of the 20 naturally-occurring amino acids, are attached (See Figure below). Peptide bonds (See Figure below) form between the carboxyl group in one amino acid and the amino group of another to form a chain designated as 1 BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report primary protein structure. Secondary protein structure forms when the chain coils in an alpha () helix or when two or more sections of the chain lie next to each other and fold into beta () pleaded sheets. Tertiary structure forms when the chain with its alpha helices and beta pleaded sheets fold back on itself. This structure is then held together by a variety of hydrogen bonds, ionic bonds, disulfide bonds, van der Waals interactions and hydrophobic interactions. If two or more proteins join, they form quaternary protein structure. The Biuret test detects proteins by reacting specifically with the peptide bonds (C-N bonds) and produces a violet color. The Biuret reagent (1% solution of copper sulfate) must complex with four to six peptide bonds to produce the purple color. Free amino acids, or very short chain peptides, are not able to turn Biuret reagent purple, but they may produce a pink color instead. Lipids Lipids are actually a heterogeneous group of substances but can generally be characterized by the fact that these molecules are hydrophobic (do not dissolve in polar solvents like water). One of the most common types of lipids is the triglyceride. This molecule consists of a glycerol backbone that is connected to three fatty acids by ester linkages. The fatty acids consist of long chains of carbons bound to hydrogen only. Lipids usually function as energy storage molecules in cells or as structural components of the membranes (phospholipids and sterols). The presence of lipids can be detected by lipid’s ability to selectively absorb pigments in fat-soluble dye, such as Sudan IV (which stains the lipid layer red). Nucleic Acids Nucleic Acids are polymers of individual nucleotide subunits joined together by phosphodiester linkages. Each nucleotide (see Figure below) consists of three parts: a pentose sugar (deoxyribose in DNA and ribose in RNA), a nitrogenous base (thymine, adenine, guanine or cytosine in DNA or uracil, adenine, guanine or cytosine in RNA) and a phosphate group. RNA consists of a single chain of nucleotides and DNA consists of two chains that coil into a double helix. The nucleic acids DNA and RNA function in information storage and transfer based on the sequence of nitrogenous bases in the molecules. 2 BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report DNA can be chemically identified by the Dische diphenylamine test. Deoxyribose reacts with diphenylamine to form a blue complex under acidic conditions. The intensity of the blue color is proportional to the concentration of DNA. Controlled Experiments A control is an experiment that is carried out to provide a basis of comparison to other experiments. In our case, controls are known solutions which we can use to make sure our experimental procedure is working. A positive control contains the variable you are attempting to detect. If your positive control reacts in the way you expect, it indicates that your experimental procedure and reagents are working correctly. A negative control does not contain the variable you are attempting to detect and in our case includes only the solvent (water). It shows what a negative result looks like so that they can be differentiated from positive results. Procedures: (work in a group of 4 students) Material on the supply bench for each group: 4 Orange racks for small test tubes 30 clean, dry small test tubes 1 small beaker (250 ml) for boiling some of the reactions 1 hot plate 2 Metal clamps for holding hot tubes in water bath. Masking tape Marker pen PARAFILM: 3 small pieces. Testing materials (onion juice, potato juice, 1% sucrose, 1% glucose, 1% starch, milk, eggwhite, BSA protein solution, 1% glycine, vegetable oil) Testing reagent (Benedict’s, Iodine, Biuret, Sudan IV, 2.5% NaOH) Demo material (total yeast RNA, calf thymus DNA, diphenylamine) 2 clean slides in blue slide box (to be reused between sections) Unknown sample for each student The Benedict’s Test For Reducing Sugars: 1. Using masking tape label a set of test tubes with A to G. 2. Add about 10 drops of each test solution to the appropriate test tube according to Table 1, then add 0.5 ml of the Benedict’s solution to each test tube. Mix sample thoroughly before transfer it to your tubes. 3. Shake the tubes gently to mix and then place them in a boiling water bath for 5 minutes. 4. Note any color changes. Any color that is not blue indicates the presence of reducing sugars. Record your results in Table 1. The Iodine Test for Starch (a polysaccharide): 1. Using masking tape label a set of test tubes with A to G (Table 1). 5. Add about 10 drops of each solution to the appropriate test tube according to Table 1. Mix sample thoroughly before transfer it to your tubes. 3 BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report 2. Add 5 drops of the Lugol’s Iodine (I2KI) solution and note any color changes. A blue to blueblack color usually indicates the presence of starch. 3. Record your results in Table 1. Table 1: Solutions and Color Reactions for Benedict’s Test and Iodine Test Benedict’s Color Reaction Iodine Color Reaction Tube Solution 10 drops of onion juice A B 10 drops of potato juice C 10 drops of 1% sucrose solution D 10 drops of 1% glucose solution (Control for benedict’s) 10 drops of 1% starch solution (control for Iodine) 10 drops of milk E F 10 drops of distilled water (Control) G The Biuret Test for the Presence of Proteins: 1. Using masking tape label a set of test tubes with H to L (Table 2). 2. Add 1 ml of each test solution to the appropriate test tube according to Table 2. 3. Add 1 ml of 2.5% sodium hydroxide (NaOH) solution to each test tube and gently mix. CAUTION: SODIUM HYDROXIDE CAN CAUSE BURNS. 4. Add 5-10 drops of the Biuret reagent (1% solution of copper sulfate (CuSO4)). 5. Observe for a color change. Violet color usually indicates the presence of proteins. 6. Record your observations in Table 2. Table 2: Solutions and Color Reactions for the Biuret Test Tube H Solution 1 ml egg white I 1 ml milk J 1 ml amino acid solution (1% glycine) K 1 ml distilled water (control) L 1 ml Protein solution (1% Bovine Serum Albumin) (Control) Color The Sudan IV Test for Lipids: 1. Label a set of test tubes with the following labels: M, N, O. 2. Add solutions according to Table 3 and shake well with the stretched Parafilm on top of the tube. When a lipid is added to water, you will observe two layers and Sudan IV should be present in the top lipid layer. 4 BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report 3. Record your results in Table 3. Table 3: Solutions and Color Reactions for the Sudan IV Test Tube M Solution 1ml water l + 5 drops of Sudan IV N 1ml water + 10 drops of vegetable oil + 5 drops of Sudan IV 1ml water + 10 drops of vegetable oil + 5 drops of detergent (Triton X-100 ) + 5 drops of Sudan IV O Description of Reaction The Dische diphenylamine Test for DNA (demonstration in chemical hood) 1. (TA) Add the material listed in Table 4 to each tube and then add 1 ml of the Dische diphenylamine. Mix thoroughly. 2. (TA) Place the tubes in a gently boiling water bath to speed up the reaction for 10 minutes. 3. (TA) Gently mix and observe the colors. (Student) Record results in Table 4. Table 4: Dische Diphenylamine Test for DNA (DEMO – observe in fume hood) Tube Solution 1 1ml calf-thymus DNA solution 3 1ml yeast total RNA solution 4 1ml water Color Unknown Identification: 1. Each student obtains a numbered tube of an unknown solution. The possibilities are: sucrose, glucose, starch, and BSA protein. Mix the sample thoroughly before you transfer it to your experiment test tubes. 2. Run the necessary tests to figure out the identity of the solution. Be sure to include positive and negative controls for your tests. Return the sample tube and dropper to TA. 3. Show your results to your TA before you clean the tubes. 4. Present your procedure (listed steps) and result (tables) clearly in the lab report. Cleaning Up: Each group must: a) Dispose of the test tubes containing vegetable oil in the broken glass box. b) Dispose of slides and slide covers in the broken glass box. c) Rinse all of the used test tubes with tap water a couple of times, SCRUB with the tube brush, rinse again, hold up to the light to make sure tube is clean, and then place the clean tubes upside down in the metal basket. Lab Report: (20 pts total): Must have a title page. Part I: Answer the following questions. Include questions 1-10 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) 5 BIOL 2281, Spring 2016 E6: Testing of Biomolecules Procedure/Report 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) 3) How can a substance taste sweet, yet give a negative reaction with the Benedict’s test? (1pt) 4) What have you learned about how carbohydrates are stored in onions and potatoes from the experiments performed in Table 1? (1 pt) 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) 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) 7) What can you conclude about the molecular content of milk based on all of the tests you performed on milk? (1 pt) 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) 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) 10) Hand-draw (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 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) 2) Write a simple summary or conclusions paragraph (4-5 sentences) stating the identity of your unknown and how you ruled out other possibilities. TO RECIEVE CREDIT YOU MUST INDICATE THE NUMBER OF YOUR UNKNOWN IN THIS PARAGRAPH. (3 pts) 6 E6: Biochemical Testing of Macromolecules The “Suspects” and Their Tests • Carbohydrates Structure and Tests – Benedict’s Test – Iodine Test • Protein Structure and Test – Biuret Test • Lipid Structure and Test – Sudan IV Test • Nucleic Acid Structure and Test – Diphenylamine Test Polymer Principle • Macromolecules (except lipids) are polymers consisting of many small repeating molecules called the monomers • • • • Carbohydrates: Polymer of monosaccharides Proteins: Polymer of amino acids Nucleic Acids: Polymer of nucleotides Lipids: Based on hydrocarbons Monosaccharides • Chemically, monosaccharides are either polyhydroxyaldehydes (aldose) or polyhydroxyketones (ketose) • Can be also classified by the number of carbons. Cyclization of aldoses and ketoses • • The carbonyl carbon of an aldose (having at least 5-C) or of a ketose (having at least 6-C) can react with an intramolecular hydroxyl group to form a cyclic form. In solution, cyclic aldoses and ketoses exist in equilibrium with noncyclic versions Dr. Pickett & Dr. Lin – Spring 2016 1 Polysaccharides--Cellulose Polysaccharides: Starch and Glycogen Benedict’s Test for Reducing Sugars Benedict’s Test Results • Benedict’s test is used for detecting reducing sugars: Boil Mixture for 3 min • Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars. • Ketoses can also be reducing sugars if they contain alphahydroxy-ketones or if they can isomerise to aldoses during the reaction. • Reducing sugars include: glucose, fructose, glyceraldehyde, arabinose, lactose, and maltose • Sucrose is not a reducing sugar (the glycosidic bond between it’s components, fructose and glucose, prevents their isomerization) Iodine Testing for Starch • Iodine interacts with coiled polymer of glucose and becomes black • Blue: - • Green: + • Yellow: ++ • Orange: +++ • Red/Brown: ++++ • Why is water blue?????? Iodine Testing for Starch • Procedure and Results – add drops of Iodine Solution (iodinepotassium iodide: I2KI) to the substance being tested – At room temperature • Bluish Black: ++ • Yellow Brown/orange: - Dr. Pickett & Dr. Lin – Spring 2016 2 Proteins: Polymers of amino acids Peptide Bonds in Proteins Biuret Test for Proteins • Cu++ react with at least 4-6 peptide bonds to produce color change to the Biuret reagent • Procedure and Result Four Levels of Protein Structure Lipids: Fats, Steroids, and Phospholipids – Mix drops of Biuret Reagent to the material and NaOH – Results at Room Temperature • Light blue: • Slight purple: + • Deep violet: ++ Dr. Pickett & Dr. Lin – Spring 2016 3 Sudan IV Test for Lipids • Sudan IV is a fat-soluble dye; will stain the lipid layer only • Emulsifier: a type of surfactant with hydrophilic and hydrophobic groups. (ex: Triton X-100) Diphenylamine Test For DNA • Acidic conditions convert deoxyribose to a molecule that binds with diphenylamine to form a blue complex Unknown Identification • Possible candidate solutions – – – – Glucose Sucrose Starch Protein • Available reagents: – Benedict’s – IKI (Lugol’s Iodine) – Biuret’s The Importance of Control Reactions • Positive Control – Shows that your test or experiment runs correctly (procedures, reagents, equipment) • Negative Control – Does not contain the variable for which you are testing; it shows you what a negative result looks like. Dr. Pickett & Dr. Lin – Spring 2016 4