Laboratory 3 Chemical Composition of Cells (LM pages 29 42) Time Estimate for Entire Lab: 2.0 hours To reduce experimental time and preparation time, the following procedures can be set up as demonstrations: Enzyme Function of Proteins (LM page 32), Starch Composition (LM page 37), and Emulsification of Lipids (LM page 39). In any case, to allow enough time for digestion, you may wish to do Enzyme Function of Proteins first, and Starch Composition immediately thereafter. Special Requirements 1. Incubation. Incubation is required for Enzyme Function of Proteins and for Starch Composition. 2. Fresh material. The potato and onion for Carbohydrates (LM page 34) and the materials for Testing the Chemical Composition of Everyday Materials and an Unknown (LM page 41) can be obtained locally, close to time of use. Seventh Edition Changes This was Lab 2 in the previous edition. New or revised figures: 3.1 Peptide bond; 3.3 Glucose; 3.4 Maltose; 3.5 Starch; 3.6 Synthesis of a fat; 3.7 Emulsification Notes 1. Test tubes. The exercises in this laboratory require students to add solutions to test tubes. As an expedient, the students are asked to mark off the tubes at various centimeter levels with a ruler and then to fill to these marks. You may prefer to have students use a standard method of measuring volume, such as with a graduated cylinder or a pipette. Most experiments use the standard size test tube. A few experiments require the large size test tube. Mini test tubes can be substituted for most laboratory exercises as long as the total volume in a given tube does not exceed 9 cm. This will reduce the volume of reagents used by approximately one-third. Test tube sizes/volumes are as follows: Mini 13 100 mm (Carolina 73-0008) 1 cm = 1.0 ml Standard 16 150 mm (Carolina 73-0014) 1 cm = 1.5 ml Medium large 20 150 mm (Carolina 73-0019) 1 cm = 2.4 ml Large 25 150 mm (Carolina 73-0025) 1 cm = 4.0 ml 2. Students results. If students results are not as expected, you may wish to test the reagents. 3. Chemical solutions. All chemicals for this laboratory can be purchased from Carolina Biological Supply. Information in the Materials and Preparations section may be helpful to those who wish to prepare their own. 4. An incubator is recommended for some tests. However, a hot water bath (hot plate, beaker of water, Celsius thermometer, test-tube clamp, beaker clamps, boiling chips) will also work. MATERIALS AND PREPARATIONS 1 All Exercises wax pencils rulers, plastic millimeter test tubes and racks water, distilled boiling chips, pumice (Carolina 84-8278, -8280)
13 3.1 Proteins (LM pages 30 33) biuret reagent (Carolina 84-8213) 1% albumin solution (Carolina 84-2250, -2252) 1 2% pepsin solution (Carolina 87-9378, -9380) 1 2% starch solution (Carolina 89-2530) 0.2% hydrochloric acid (HCl) (Carolina 86-7790, -7791) incubator set at 37 C or water bath Biuret reagent. 30 ml per student group should be sufficient (using standard test tubes for all procedures). If you buy prepared biuret, use only ten to fifteen drops; otherwise, the solution will be too dark, or dilute to a 10% solution (10 ml biuret with 90 ml distilled water). To prepare your own biuret reagent, maintain separate stock solutions of 3% copper sulfate 3 g of copper sulfate (cupric sulfate, Carolina 85-6550) per 100 ml of distilled water and 10% potassium hydroxide or sodium hydroxide 100 g of potassium hydroxide (Carolina 88-3488) or sodium hydroxide pellets (Carolina 88-9470) per 1,000 ml of distilled water). Adding five drops of copper sulfate solution and ten drops of potassium hydroxide solution to each experimental tube produces more consistent results. Biuret reagent should be prepared fresh for each lab. 1% albumin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Prepare ph 7 buffer solution (Carolina 84-9380) as per directions on the vial, and dissolve 1 g of albumin per 100 ml of buffer. Allow time for precipitation to occur, and then decant. Also, check ph with indicator paper, and adjust to ph 7 with dilute acid or base. Swirl the stock, before distributing it to students. 1 2% pepsin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). To make a 1% solution, dissolve 1 g of pepsin in 100 ml of distilled water. Stir to dissolve. 1 2% starch solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Care must be taken in preparing this solution. To make a 1% solution, dissolve 1 g of starch in a small amount of cold water to form a pourable paste. Add this to 100 ml of boiling distilled water, while stirring, and mix a few minutes. Cool. Add a pinch of sodium chloride (NaCl). If refrigerated, this solution will last for several weeks; otherwise, a fresh supply should be prepared each day. 0.2% hydrochloric acid (HCl). 20 ml per student group should be sufficient (using standard test tubes for all procedures). Add 2.3 ml of concentrated HCl to 100 ml of distilled water. 3.2 Carbohydrates (LM pages 34 37) 1% starch solution (Carolina 89-2530), see prep notes above iodine-potassium-iodide (IKI) solution, premade (Carolina 86-9051, -9053, -9055), or see Laboratory 2 for preparation instructions from potassium iodide (KI) and iodine (I) crystals dropper bottles, one per student group (Carolina 71-6525) razor blades, single-edged cutting board for potato, onion potato, fresh microscopes, compound light lens paper slides coverslips onion, fresh paper towels boiling water bath: hot plate large beakers boiling chips, pumice (Carolina 84-8278, -82800 beaker clamps thermometer, organic based, 50 150 C range (optional) 1% glucose (dextrose) solution (Carolina 85-7430) onion juice from fresh onion 1 Note: Materials and Preparations instructions are grouped by exercise. Some materials may be used in more than one exercise.
14 potato juice from fresh potato mortar and pestle Benedict s reagent (Carolina 84-7091, 84-7111) 1% pepsin solution (Carolina 87-9378, -9380) 0.2% hydrochloric acid (HCl) (Carolina 86-7790, -7791) 1% amylase solution (Carolina 20-2350) incubator at 37 C or hot water bath Iodine (IKI) solution. Use one dropper bottle per student group. For ease of comparison, the same amount should be used each time. Premade iodine-potassium-iodide solution can be purchased, or the ingredients can be purchased separately as potassium iodide (KI) (Carolina 88-3790,-3792) and iodine (I) (Carolina 86-8970,-8972). These dry ingredients have a long shelf life and can be mixed as needed, according to the instructions in Laboratory 2. Boiling water bath. Place a large beaker of water on a hot plate. Adjust the dial on the hot plate so that the water is maintained at a gentle rolling boil during the experiment. Thermometers are optional, since students should know that boiling water is 100 C. 1% glucose solution. This makes enough for all procedures for 20 student groups. Place 1 g of glucose in 50 ml of distilled water. Stir to dissolve, and bring the volume up to 100 ml. Onion and potato juice. Onion may be mashed the day before, then stored in a refrigerator overnight. Potato juice should be extracted as close to time of use as possible. Benedict s reagent. 50 ml per student group is sufficient. Benedict s reagent can be purchased as a powder to make 1 liter. Or to make 1 liter, mix 173 g of sodium citrate (Carolina 88-9060) and 100 g of sodium carbonate, anhydrous (Na 2 CO 3 ) (Carolina 88-8770) with 800 ml of distilled water. Warm this mixture to dissolve; then cool and filter it. Add distilled water to make 850 ml. Then dissolve 17.3 g of copper sulfate (cupric sulfate, Carolina 85-6550) in 100 ml of distilled water, and stir slowly into the first solution. Add distilled water to make 1 liter. When testing, Benedict s reagent should be boiled approximately 5 minutes or longer. 1% amylase solution. 20 ml per student group is sufficient. Mix 1 g alpha amylase with 100 ml distilled water. Keep the alpha amylase powder refrigerated when not in use, and make the solution fresh when needed. 3.3 Lipids (LM pages 38 40) paper, brown (squares 4 cm 4 cm) water vegetable oil test tubes Tween (Carolina 20-7861, -7862) or bile salts (oxgall, Wards Biology 38W2179) medicine droppers, dropper bottles slides coverslips microscopes, compound light lens paper adipose tissue slide (Carolina 31-2698, -2728) everyday materials for unknowns; suggestions: bread crumbs, flour and water mixture, squeeze bottle of margarine, skim milk, orange juice, syrup from canned fruit 1% Tween or bile solution. Tween is a standard wetting agent. Measure 1 ml of Tween or 1 g of bile salts, and dissolve in 100 ml of distilled water.
15 EXERCISE QUESTIONS 3.1 Proteins (LM page 30) Test for Proteins (LM page 30) Experimental Procedure: Test for Proteins (LM page 31) Table 3.2 Biuret Test Tube Contents Final Color Conclusions 1 Distilled water Blue No protein is present. 2 Albumin Purple Protein is present. 3 Pepsin Purple Protein is present. 4 Starch Blue No protein is present. Conclusions (LM page 32) If your results are not as expected, offer an explanation. Answers will vary. Which of the four tubes is the control sample? tube 1 What is a control sample? In a control sample, the sample does not contain the factor being tested. Therefore, a control sample is expected to give negative results. Why do experimental procedures include control samples? The control samples usually validate the experiment. If control samples give positive results, the experiment is invalid in this case, the reagents may be contaminated or the procedure may need improvement. Experimental Procedure: Enzyme Function of Proteins (LM page 32) Table 3.3 Action of Pepsin Tube Contents Final Color Conclusions 1 Albumin Water Purple The protein albumin is present. HCl* (incubate) 2 Albumin Pepsin Pinkish-purple to pink Albumin is digested to peptides by HCl (incubate) the enzyme pepsin. *HCl may cause some breakdown. Conclusions (LM page 33) Why were the tubes incubated at 37 C rather than at another temperature? This is normal human body temperature, the optimum for digestion. Enzymes are specific and speed up only one type of chemical reaction. Considering this, do you predict that pepsin will break down starch? no Why or why not? Starch does not contain peptide bonds, which are specifically broken by pepsin. If your results are not as expected, offer an explanation. Answers will vary.
16 3.2 Carbohydrates (LM page 34) Test for Starch (LM page 35) Experimental Procedure: Test for Starch (LM page 35) Potato (LM page 35) 3. What is the color of the small oval bodies? blue-black What is the chemical composition of these oval bodies? starch Onion (LM page 35) Table 3.4 Iodine (IKI) Tests for Starch Sample Color Change Conclusions Tube 1: Starch Blue-black Starch is present. Tube 2: Water Orange-brown (no change) No starch is present. Potato Blue-black Starch is present. Onion Orange-brown (no change) No starch is present. Conclusions (LM page 35-36) Is glucose stored as starch in the potato? yes Is glucose stored as starch in the onion? no Test for Sugars (LM page 36) Experimental Procedure: Test for Sugars (LM page 36) Table 3.6 Benedict s Reagent Test Tube Contents Color (After Heating) Conclusions 1 Water Blue No sugar is present. 2 Glucose solution Orange A very high amount of sugar is present. 3 Onion juice Yellow to orange A moderate amount of sugar (as per Table 3.5) is present. 4 Potato juice Yellow Some sugar is present. 5 Starch suspension Usually blue; may be yellow No sugar to some sugar is present. Conclusions (LM page 37) From your test results, conclude what kind of chemical is present and why the results occurred. Enter your conclusions in Table 3.6. Which tube served as a control? tube 1 Starch Composition (LM page 37) Experimental Procedure: Starch Composition (LM page 37) Table 3.7 Starch Composition Tube Color Change Conclusions 1 Water None No sugar is present. Amylase 2 Starch Green Maltose is present. Amylase Conclusions (LM page 38) From your test results, you may conclude that starch is composed of what kind of chemical? sugar
17 3.3 Lipids (LM page 38) Test for Lipids (LM page 38) Experimental Procedure: Test for Lipids (LM page 38) 1. Place a small drop of water on a square of brown paper. Describe the immediate effect. The water sits on top may evaporate away. 2. Place a small drop of vegetable oil on a square of brown paper. Describe the immediate effect. Oil soaks into the paper and creates an oil spot. Table 3.8 Test for Lipids Sample Results Conclusions Water spot Does not penetrate paper Substance is not an oil. Oil spot Penetrates paper Substance is an oil. Emulsification of Lipids (LM page 39) Experimental Procedure: Emulsification of Lipids (LM page 39) Tube 1: 2. Is vegetable oil soluble in water? no Tube 2: 2. Describe how the distribution of oil in tube 2 compares with the distribution in tube 1. There is no longer a separation into two separate layers. The oil is distributed throughout the tube. Microscopic Examination (LM page 40) Table 3.9 Emulsification Observations (Microscopic) Tube Contents Observations Conclusions 1 Oil If any oil droplets are visible, they are large. Oil does not mix with water. Water 2 Oil Oil droplets are numerous and small, Emulsifier causes oil to mix with water. Water due to the effect of the emulsifier. Emulsifier Conclusions (LM page 40) Explain the correlation between your macroscopic observations (how the tubes look to your unaided eye) and your microscopic observations. The cloudy layer in tube 2 is due to suspended, small oil droplets. 3.4 Testing the Chemical Composition of Everyday Materials and an Unknown (LM page 41) Experimental Procedure: Chemical Composition (LM page 41) 3. Record your result as positive or negative in Table 3.10. Results will vary depending on the material provided by the instructor. Conclusions (LM page 41) Was there any material that tested positive for only one of the organic compounds? e.g., sucrose Explain. Sucrose is a pure carbohydrate. What types of foods would you expect to test positive for more than one of the organic compounds studied in this laboratory? e.g., milk has carbohydrate, fat, and protein in it
18 LABORATORY REVIEW 3 (LM page 42) 1. What type of bond joins amino acids to make peptides? peptide 2. What type of protein speeds chemical reactions? enzyme 3. What is NH 2? amino group 4. If iodine (IKI) solution turns blue-black, what substance is present? starch 5. If Benedict s agent turns red, what substance is present? glucose 6. What is the function of starch in plant cells? stored energy 7. What is starch composed of? sugar 8. What is the function of fat in animal cells? stored energy 9. What molecules are released when fat is hydrolyzed? glycerol, fatty acids 10. What can be added to make fat disperse in water? emulsifier 11. Are fats polar or nonpolar? nonpolar 12. If biuret reagent turns purple, what substance is present? protein movement 13. A student adds iodine to egg white and waits for a color change. How long will the student have to wait? forever 14. To test whether a sample contains glucose, what reagent should be used? Benedict s Thought Questions 15. Why is it necessary to shake a abottle of salad dressing before adding it to a salad? The oil in salad dressing is not soluble in water, and shaking mixes the otherwise separate layers. 16. An unknown sample was tested with both biuret reagent and Benedict s reagent. Both tests result in a blue color. What has been learned? No protein and no sugar were present.