1Amino Acids, Peptides, and Proteins Introduction Amino Acids Amino acids are the building blocks of proteins. In class you learned the structures of the 20 common amino acids that make up proteins. All amino acids have the general structure shown below. H O H 3N + C C O R Peptides Peptides are short chains of amino acids, each one connected to the next by an amide linkage called a peptide bond. Below is the chemical reaction by which two amino acids become connected by an amide linkage (the circled O and two H‟s are eliminated as a water molecule during the reaction): H O H3N C C O R + - H H O + H N C C O H R + + H O H3N C C R H O N C C OH R + H2O peptide bond A tripeptide (composed of three amino acids): H O H O H O H3N+ C C R N C C H R two peptide bonds N C C OH R The artificial sweetener aspartame (brand name Nutrasweet) is an example of a modified peptide. The structure of aspartame is shown below. H O H3N C C CH2 O C O+ H O N C C O CH3 H CH2 Very small proteins may be composed of 50 to 100 amino acids. it will not react with the Biuret reagent. resulting in the formation of a violet/purple-colored product. They act as microscopic cellular machines that function in much the same manner as human-built machines. When a compound does not have at least two peptide bonds. and no purple color will appear (solution will remain a shade of blue due to the copper ions). resulting in the formation of a violet/purple-colored product. In the denaturation test. Protein molecules are long chains of amino acids connected to each other in the same manner as in peptides. and proteins in the lab. If no precipitate forms. strong acid is used to test a solution for the presence of protein.2 Proteins Proteins are the work horses of living cells. When a compound does not have a primary amino group. it will not react with the ninhydrin reagent. proteins that have been denatured tend to clump together and come out of solution as a precipitate because they are no longer soluble when they are denatured and clumped together. the protein will no longer be able to carry out its biological function and will usually be destroyed by the cell. Biuret Test The Biuret reagent contains copper ions which give it a blue color. Positive Biuret test: violet/purple product forms Negative Biuret test: no violet/purple product formed Ninhydrin Test The ninhydrin reagent will react specifically with a primary (1o) amino functional group on a compound. and no purple color will appear (solution will remain colorless). If the 3-dimensional shape “unfolds” for some reason. Chemical Tests In this experiment you will perform three chemical tests to distinguish between free amino acids. Every protein has a unique 3-dimensional shape that is suited to its biological function in a living organism. while large proteins may contain thousands of amino acids. The copper ions will interact with a compound that contains two or more peptide bonds.) The unfolding of a protein structure is called denaturation. Positive ninhydrin test: violet/purple product forms Negative ninhydrin test: no violet/purple product formed Denaturation Test Strong acid will often denature (unfold) proteins. peptides. the denaturation test is negative. However. . the long chain of amino acids is “folded” into a 3dimensional shape (imagine the long amino acid chain as a piece of string that has been crumpled up into a 3-D ball/blob). Proteins that are properly folded into their normal 3-dimensional shape tend to be soluble in aqueous solution. and indicates that no protein is present in the solution. In a normal functional protein. (Unfolding is like the crumpled up blob of string being unwound again. the formation of a white precipitate (composed of denatured protein molecules) is considered a positive denaturation test and indicates the presence of protein in the solution. When strong acid is added. Indicate whether each will give a positive or negative Biuret test. and Proteins Prelab Name_______________ 1. or a protein.k. A.) ___________ ___________ ___________ 4. A. Biuret test? (+ or . Indicate whether each of the following is an amino acid. Examine the structure of aspartame (Nutrasweet) in the introduction.a. aspartate) ____________ phenylalanine Nutrasweet (aspartame) ____________ ____________ B. pept. a peptide. paying careful attention to the side chains. Which two amino acids would be released if the peptide bond was „hydrolyzed‟ (broken by a reaction with H2O)? .3 Amino Acids. Draw the structures of alanine and glycine. B. 3. Show the structure of the peptide that would result from a chemical reaction between glycine and alanine. Peptides. (Refer to the figure showing the 20 amino acids which is included in your In-Class Biochemistry Notebook.) 2. prot? aspartic acid (a. aa. Add acetic acid until you see no further precipitation forming.1 M NaOH to the casein and stir to dissolve. If you wish. the milk protein casein becomes insoluble and begins to precipitate. It is very important that the test tubes be clean in order to prevent anomalous results in the chemical tests you will carry out. While stirring gently. Biuret Test 1. you may also do step 1 of the ninhydrin test below while you are waiting. . but shake them to remove as much water as possible. Allow the contents to stand for 10 minutes. Tube 5: 1 mL of the unknown Then add 1 mL of the biuret solution to each of the five test tubes. This is the solution of dissolved casein you will use in the following steps. and Proteins Procedure Isolation of the Protein Casein from Milk 1. 4. Put 50 mL of skim milk into a 250 mL beaker. Wash the casein with 25 mL of ethanol. Prepare a simple filtration apparatus as follows. Pour your precipitated casein mixture through the cheesecloth. 2. 2. add a solution of 10% acetic acid dropwise to the milk. While waiting. NOTE: glycylglycine is a dipeptide composed of two glycine‟s linked together. Place a square of cheese cloth (about 3 layers thick) over the top of a large beaker—the cheesecloth square should be just large enough to cover the top of the beaker with a little overlap at the edges. Scrape the casein off the cheesecloth and press it between some paper towels to dry it. Secure the cheesecloth using a rubber band. but don‟t turn on the hot plate yet (you‟ll need it for the ninhydrin test below). fill a 250 mL beaker about 1/3 full with distilled water and place it on a hot plate.5). 3. Start with 5 drops and show your results to the instructor to see if this is enough.4 Amino Acids. Add 10 mL of 0. Rinse them well with distilled water. The test tubes do not have to be completely dry. Take about half of your casein and put it in a 50 mL beaker. As the mixture becomes acidic (at approximately pH 5. Peptides. Place the following chemicals into four test tubes as indicated: Tube 1: 1 mL distilled water (water will serve as a control) Tube 2: 1 mL 1% alanine solution Tube 3: 1mL 1% glycylglycine solution Tube 4: 5 drops of your casein solution (from step 4 above) + 1 mL distilled water NOTE: It may take more than 5 drops of the casein. Use the dilute soap solution in the bottles near the sinks (bottles with blue lids) to thoroughly clean 5 test tubes. At the end of 5 minutes. Answer the questions regarding interpretation of the test results. place the tubes in the boiling water bath for 5 minutes—be sure that the water level is not so high that water boils over into the test tubes. observe and record the color of the test tube contents on the report sheet. and test the pH again. The contents of the test tubes can then be discarded down the drain. Use more drops of HCl or NaOH as needed to achieve a pH of 7 to 8. but it needs to be neutral (pH ≈ 7) to conduct a ninhydrin test. 2. Turn the hot plate on high to start heating the water—you will need boiling water for the ninhydrin test. Place the following chemicals into the five clean test tubes: Tube 1: 1 mL distilled water (water will serve as a control) Tube 2: 1 mL 1% alanine solution Tube 3: 1mL 1% glycylglycine solution Tube 4: 1 mL of neutralized casein solution from step 1 directly above Tube 5: 1 mL of the unknown Then add 1 mL of 0. and test the pH again. Put 3 drops of the casein solution from step 4 above into a clean test tube and add 1 mL of distilled water.3 M NaOH to the test tube. At the end of 10 minutes. mix the contents of the tube. Answer the questions regarding interpretation of the test results. Ninhydrin Test 1. Test the pH of the solution by dipping a glass stirring rod into the solution and then touching the end of the rod onto a piece of pH paper. add a drop of 0. 4. Label the tubes clearly using a grease pencil. Observe and record the color of the tube contents on the report sheet.5 3. . If the pH is too high (above 7-8). 3. add a drop of 0. Once the water is boiling moderately. Your casein solution is acidic (pH below 7). They need not be completely dry. Don‟t use tape to label the tubes because the tape will fall off in the boiling water bath in the next step. Wash the five test tubes from the Biuret test thoroughly so they can be re-used now.2% ninhydrin solution to each test tube. Neutralize your casein solution as follows. The contents of the test tubes can then be discarded down the drain. If the pH is too low (below 7-8).3 M HCl to the test tube. turn off the hot plate. mix the contents of the tube. ) Answer the questions regarding interpretation of the test results. Observe and record your observations on the report sheet. 3. NOTE: if you see purple color in any of the solutions. the test tube was not thoroughly cleaned after the ninhydrin test. The contents of the test tubes can then be discarded down the drain. (There may be nothing to observe—no changes—in some of the tubes. The solid casein can be discarded in the trash.6 Denaturation with Acid 1. Mix the contents of the tubes by flicking them with your finger. Be sure to use a test tube brush and soap in order to remove any purple residue from the ninhydrin test. All other solutions can be discarded down the drain. The tubes need not be completely dry. Place the following chemicals into the five clean test tubes: Tube 1: 1 mL distilled water (water will serve as a control) Tube 2: 1 mL 1% alanine solution Tube 3: 1mL 1% glycylglycine solution Tube 4: 10 drops of your solution of dissolved casein in the 50 mL beaker from step 4 under “Isolation of the Protein Casein from Milk” Tube 5: 1 mL of the unknown Then add 1 mL of 20% trichloroacetic acid to each tube. Wash the five test tubes from the ninhydrin test thoroughly so they can be re-used now. 2. . #2) in order to interpret the results of the test. and Proteins Biuret Test water (control) 1% alanine 1% glycylglycine casein unknown Color observed Report Sheet Name ___________ Interpretation of Biuret Test Results It will be helpful to review the structures of alanine and glycylglycine (see Postlab #1.7 Amino Acids. Peptides. What do the results of the Biuret test tell you about alanine? What do the results of the Biuret test tell you about glycylglycine? What do the results of the Biuret test tell you about casein? Ninhydrin Test water (control) 1% alanine 1% glycylglycine casein unknown Color observed . What do the results of the ninhydrin test tell you about alanine? What do the results of the ninhydrin test tell you about glycylglycine? What do the results of the ninhydrin test tell you about casein? Denaturation Test water (control) Observations 1% alanine 1% glycylglycine casein unknown Interpretation of Denaturation Test Results What do the results of the denaturation test tell you about alanine? What do the results of the denaturation test tell you about glycylglycine? What do the results of the denaturation test tell you about casein? .8 Amino Acids. #2) in order to interpret the results of the test. Peptides. and Proteins Report Sheet Name ___________ Interpretation of Ninhydrin Test Results It will be helpful to review the structures of alanine and glycylglycine (see Postlab #1. Use an arrow and a brief description to indicate the structural feature that caused the glycylglycine solution to produce the Biuret test result that it did.9 Amino Acids. Draw the structure of alanine. Oxytocin is a hormone. Predict the results (+ or -) of the following tests on hemoglobin: Biuret test:___ Ninhydrin test:___ Denaturation test:___ 6. Look back at the test results you obtained for the uknown. Peptides. Predict the results (+ or -) of the following tests on aspartame: Biuret test:___ Ninhydrin test:___ Denaturation test:___ 4. What tests could you use to determine which is in the test tube? . 2. Draw the structure of glycylglycine (first refer to the “NOTE” on step 2 on page 4). Look at the structure of aspartame (Nutrasweet) in the introduction. check the identity of your unknown. and Proteins Postlab Name_______________ 1. Hemoglobin is a protein composed of over 100 amino acids linked together by peptide bonds. You have a test tube containing either a dipeptide or a large protein. What was the purpose of having water as a control for each test? 8. Circle the structural feature that caused the alanine solution to produce the ninhydrin test result that it did. Predict the results (+ or -) of the following tests on oxytocin: Biuret test:___ Ninhydrin test:___ Denaturation test:___ 5. 3. Based on the test results. it is a 9-amino acid peptide. An amino acid ___ A pentapeptide (composed of 5 amino acids) ___ A protein (composed of several hundred amino acids) ___ 7.