(LM pages 91 98) Time Estimate for Entire Lab: 2.5 to 3.0 hours. Special Requirements

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1 Laboratory 7 Chemical Aspects of Digestion (LM pages 91 98) Time Estimate for Entire Lab: 2.5 to 3.0 hours Special Requirements Incubation. Students should start these sections at the beginning of the laboratory: 30-minute incubations for Experimental Procedure: Digestion (LM page 92); two-hour incubations for Experimental Procedure: Protein Digestion (LM page 96). Seventh Edition Changes This was lab 6 in the previous edition. Experimental Procedure: Fat Digestion was revised. It now uses vegetable oil as the fat, instead of litmus cream. MATERIALS AND PREPARATIONS 1 All Exercises wax pencils test tubes and racks water, distilled thermometer, celsius (0 110 C) rulers, plastic millimeter Demonstrations. If the laboratory is short, you may wish to incubate the digestion tubes overnight and demonstrate the experiments. Enzyme solutions. Enzyme solutions must be free of granules. If desired, a solution can be prepared at the beginning of the week and kept in the refrigerator for multiple laboratory sessions. If this is done, warm the experimental test tubes to room temperature in a water bath before incubating. To make a 1% enzyme solution, dissolve 1 g of the enzyme powder in 100 ml of distilled water. 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 mm (Carolina ) 1 cm = 1.0 ml Standard mm (Carolina ) 1 cm = 1.5 ml Medium large mm (Carolina ) 1 cm = 2.4 ml Large mm (Carolina ) 1 cm = 4.0 ml 1 Note: Materials and Preparations instructions are grouped by exercise. Some materials may be used in more than one exercise.

2 Digestion by Salivary Amylase (LM pages 92-93) amylase solution (Carolina ) boiling water bath test-tube holder hot plate beaker beaker tongs starch solution (Carolina ) iodine-potassium-iodide (IKI) solution, premade (Carolina , -9053, -9055) Benedict s reagent powder (Carolina ) or Benedict s reagent solution (Carolina , -7113) Amylase solution. Prepare 20 ml per student group. Dissolve 1 g alpha amylase in 100 ml distilled water. solution. Prepare 20 ml per student group. A fresh supply of this solution must be carefully prepared every day. To make a 1% starch solution, dissolve 1 g of starch in a small amount of cold water to form a paste. Add this to 100 ml of boiling distilled water, and mix a few minutes. Cool. Add a pinch of sodium chloride (NaCl). Iodine (IKI) solution. Prepare 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 , -3792) and iodine (I) (Carolina , -8972). These dry ingredients have a long shelf life and can be mixed as needed, according to the instructions in Laboratory Fat Digestion by Pancreatic Lipase (LM pages 94-95) pancreatin (pancreatic lipase) (Carolina ) vegetable oil, preferably canola, olive, or sunflower phenol red solution (Carolina to ) incubator, 37 C bile salts (oxgall, Wards Biology 38W 2179). One gram is enough for a class. Pancreatin solution (1% pancreatin in 0.1% Na 2 CO 3 ). Prepare 30 ml per student group. Add 1 g pancreatin to every 100 ml of 0.1% Na 2 CO 3 (0.1g Na 2 CO 3 per 100 ml of distilled water). solution. Prepare 20 ml per student group. Use a 0.04% solution. Dissolve 0.04 g of phenol red in 100 ml of distilled water. Students should not vigorously shake tubes. Oil will float above phenol red solution and pancreatin, and a color change may be observed in the transition zone; however, it will not be uniform unless bile salts are added. 7.3 Protein Digestion by (LM pages 95-96) 1 2% pepsin solution (Carolina , -9380) incubator, 37 C albumin solution (Carolina , -2252) 0.2% hydrochloric acid (HCl) (Carolina ,-7791) biuret reagent (Carolina ) 1 2% pepsin solution. Prepare 20 ml per student group. To make a 1% solution, dissolve 1 g of pepsin in 100 ml of distilled water. Albumin solution. Prepare 10 ml per student group. Mix in a ph 7 buffer solution (Carolina ) as per directions on the vial, and dissolve 1 g per 100 ml of water. Allow time for precipitation to occur, and then decant. Swirl the stock prior to distribution to students. Also, check ph with indicator paper, and adjust to ph 7 with dilute acid or base. 0.2% hydrochloric acid (HCl). Add 0.57 ml of concentrated HCl to 100 ml of distilled water.

3 34 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 ) per 100 ml of distilled water and 10% potassium hydroxide or sodium hydroxide 100 g of potassium hydroxide (Carolina ) or sodium hydroxide pellets (Carolina ) 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. EXERCISE QUESTIONS 7.1 Digestion by Salivary Amylase (LM pages 92-93) 1. Why is this reaction called a hydrolytic reaction? breaks down as it reacts with water. 2. If digestion does not occur, which will be present starch or maltose? starch 3. If digestion does occur, which will be present starch or maltose? maltose Tests for Digestion (LM page 92) 2. To which category of organic compounds (lipid, carbohydrate, or protein) do enzymes, such as amylase, belong? proteins What happens when enzymes are boiled? Enzymes, being proteins, are denatured, or destroyed when boiled. Experimental Procedure: Digestion (LM page 92) Table 7.1 Digestion by Amylase* Tube Contents Time Type Results Explanation of Test 1 Alpha-amylase 0 (iodine) Positive: black No digestion; not enough time 2 Alpha-amylase 0 Sugar (Benedict s) Negative: blue No digestion; not enough time 3 Alpha-amylase 30 (iodine) Negative: Digestion; enough time elapsed dark purple 4 Alpha-amylase 30 Sugar (Benedict s) Positive: Digestion; enough time elapsed green-orange 5a Alpha-amylase, 30 Sugar (Benedict s) Negative: blue No digestion; enzyme is denatured boiled 5b Alpha-amylase, 30 (iodine) Positive: black No digestion; enzyme is denatured boiled 6 Water 30 Sugar (Benedict s) Negative: blue No digestion; control (no enzyme) *Results may vary somewhat, depending on enzyme purity, etc. Conclusions (LM page 93) What is the function of a control? A control shows negative results and helps eliminate errors in interpreting experimental results. Which test tube or tubes served as controls in this experiment? tube 6 Explain. Tube 6 does not contain the enzyme alpha-amylase.

4 Fat Digestion by Pancreatic Lipase (LM pages 94-95) Given the second reaction, would the ph of the solution be lower before or after the reaction? The ph would be lower after the reaction. Test for Fat Digestion (LM page 94) Experimental Procedure: Fat Digestion (LM page 94) Table 7.2 Fat Digestion by Pancreatic Lipase* Tube Contents Time Color Change Explanation Initial Final 1 Vegetable oil * Red Red No digestion (no enzyme); control Bile salts 2 Vegetable oil * Red Pink (red Limited digestion; no emulsifier at border) Pancreatin 3 Vegetable oil * Red Yellow Digestion; enzyme and emulsifier present Pancreatin Bile salts 4 Vegetable oil * Red Red No digestion (no enzyme or emulsifier); control Distilled water * *Time and overall results may vary somewhat, depending on enzyme purity, etc. Conclusions (LM page 95) What role did bile play in this experiment? Bile acts as an emulsifier and breaks large drops of fat into very small droplets. This makes more fat molecules available for digestion. What role did phenol red play in this experiment? was a ph indicator. Which two test tubes in this experiment could be considered controls? tubes 1 and 4, because they do not contain enzyme, the variable being tested 7.3 Protein Digestion by (LM pages 95-96) The stomach has a very low ph. Does this indicate that pepsin works effectively in an acidic or basic environment? acidic Test for Protein Digestion (LM page 96) Experimental Procedure: Protein Digestion (LM page 96) Table 7.3 Protein Digestion by Tube Contents Temperature Results Explanation of Test 1 Distilled water 37 C Purple No digestion (no enzyme); control Albumin 2 Albumin 37 C Purple No digestion; incorrect ph Distilled water 3 Albumin 37 C Pinkish-purple Digestion; enzyme and correct ph HCl 4 Albumin 22 C Light purple Some digestion; temperature is low to pale HCl pinkish-purple

5 36 Conclusions (LM page 96) Which tube was the control? tube 1 Explain. Tube 1 contained no enzyme (pepsin). If this control tube had given a positive result for protein digestion, what could you conclude about this experiment? The experiment is invalid. Either protein degrades on its own, or the biuret reagent is not testing properly. Requirements for Digestion (LM page 97) Table 7.4 Requirements for Digestion Requirement Specific enzyme Specific substrate Warm temperature Time Specific ph Fat emulsifier Explanation Each enzyme speeds only one type of reaction. Enzyme and substrate must come together like a lock and key for reaction to occur. Chemical reactions occur at a faster rate at warm temperatures than at cold temperatures. It takes time for the enzyme to bring about the reaction. Optimum ph maintains the shape of the enzyme so that the enzyme will combine with its substrate. Fats are insoluble in water. The emulsifier breaks up fat so that fat droplets are exposed to the enzyme. LABORATORY REVIEW 7 (LM page 98) 1. When iodine (IKI) solution turns blue-black, what substance is present? starch 2. What color is Benedict s reagent originally? blue 3. What happens to an enzyme when it is boiled? denatured 4. Saliva contains what enzyme? amylase 5. As oil is digested, why does the first tube turn from red to yellow? Fatty acids are released. 6. What temperature promotes enzymatic action? body temperature 7. What do you call a sample that goes through all the steps of an experiment, but lacks the factor being tested? control 8. What role do bile salts play in digestion of fat? an emulsifier 9. What color does biuret reagent turn when peptides are present? pinkish-purple 10. Is the optimal ph for pepsin acidic or basic? acidic 11. Why would you predict that pepsin would not digest starch? Enzymes are specific. 12. In addition to pepsin and water, what is needed to digest protein? HCl Thought Questions 13. Which of the following two combinations is most likely to result in digestion? a., protein, water, body temperature b., protein, hydrochloric acid (HCl), body temperature Explain. The second combination (pepsin, protein, HCl, body temperature) is more likely to result in digestion because the optimum ph for pepsin is acidic. 14. Which of the following two combinations is most likely to result in digestion? a. Amylase, starch, water, body temperature, testing immediately b. Amylase, starch, water, body temperature, waiting 30 minutes Explain. The second combination (amylase, starch, water, body temperature, waiting 30 minutes) is more likely to result in digestion because there is more time.

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