Chemical and Physical Processes of Digestion Exercise 39A / 39 (begins page 597 in 9 th &10 th eds, page 595 in 11 th edition, page 599 in 12 th edition) Lab 7 Objectives Read lab Exercise 39A / 39 Do Activities 1 & 3 Work in groups of 3 to 4 students (enough materials for 5 groups total) ***Read through the whole lab exercise from the book including experimental directions and descriptions, but follow the directions below to set up the experiments. These instructions are similar to the book but are provided here to unify the class as the different editions do things slightly different from each other!!! All water-baths for incubation (37 C, 0 C) and boiling (100 C) are located on the side of the room for use by all groups. gtt = gutta, Latin for drop Do not put any wastes down the sink! All materials will be collected as-is at the end of class. SET UP: Activity 1: Assessing Starch Digestion by Salivary Amylase 1. Label 6 tubes by writing 1A, 2A, 3A, 4A, 5A and 6A on the glass with China marker. Then wrap your group s color of tape as high around the top of each tube as possible. Wrap the tape completely around the circumference of the tube and stick the ends of the tape together so it won t steam/boil off. 2. Add the acid to tube 4A FIRST, then add each of all the other reagents to the all tubes according to the chart below. 1A 2A 3A 4A 5A 6A Amylase 10 gtt - - 10 gtt 10 gtt 10 gtt Starch - 10 gtt - 10 gtt 10 gtt 10 gtt Maltose - - 10 gtt - - - Water 10 gtt 10 gtt 10 gtt - - - HCl - - - 3 gtt - - Temperature 37 C 37 C 37 C 37 C 37 C 0 C 3. Place all the tubes in the proper water-bath temperature for incubation according to the chart above, and incubate for 40 minutes. Activity 3: Demonstrating the Emulsification Action of Bile and Assessing Fat Digestion by Lipase 1. Label 7 tubes by writing 1L, 2L, 3L, 4L, 5L, 4B and 5B on the glass with China marker. Then wrap your group s color of tape as high around the top of each tube as possible. Wrap the tape completely around the circumference of the tube and stick the ends of the tape together so it won t steam/boil off. Amy Warenda Czura, Ph.D. 1 SCCC BIO132 Lab 7 Chemical Digestion
2. First add 10 drops of pancreatin/lipase to tube 3L, and boil tube 3L with lipase in the 100 C water-bath for 15 minutes. Then add each of all the other reagents to the all tubes according to the chart below while you are waiting. A pinch of bile salts is what fits on the pointed end of a small spatula. Be sure to use only a pinch (tiny amount) of bile salts in tubes 4B and 5B as too much will obscure the results. 1L 2L 3L 4L 5L 4B 5B Lipase 10gtt - 10gtt (BOIL) 10gtt 10gtt 10gtt 10gtt Litmus 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt Cream - 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt Water 10gtt 10gtt - - - - - Bile salts - - - - - Pinch Pinch Temperature 37 C 37 C 37 C 37 C 0 C 37 C 0 C 3. After Tube 3L with lipase has boiled for 15 minutes, add 10 drops each of litmus and cream. 4. After all the reagents are in the tubes, place a square of parafilm over the top of tubes 4B and 5B and vortex for 30 second to mix the contents thoroughly. 5. Place all the tubes in the proper water-bath temperature for incubation according to the chart above, and incubate for 1 hour. AFTER INCUBATION (40 min for amylase, 60 min for lipase): Activity 1: Assessing Starch Digestion by Salivary Amylase 1. Label 6 wells of the spot plate for tubes 1A-6A with the China marker and transfer 1 drop from each tube into the appropriate well. Add one drop of Lugol s IKI to each sample in the spot plate and record the results. 2. Into the remaining solution in each tube, add 3 drops of Benedict s reagent and boil the tubes for 5 minutes before recording the results. Activity 3: Demonstrating the Emulsification Action of Bile and Assessing Fat Digestion by Lipase 1. Record the color of the result for each tube 1L -5L, 4B, & 5B. Indicate gradations or shades in your results, such as one tube being more pink, or more purple, or more blue than another. 2. After recording all your results, perform a positive control for litmus turning pink in the presence of acid: add 3 drops of HCl to tube 2L. Do not put any wastes down the sink! Remove all tape at the conclusion of the experiment. Place all materials where directed at the end of class. ***You are responsible for your understanding of the theory, enzymatic reactions and results of these experiments, so be sure to ask if something is unclear.*** Amy Warenda Czura, Ph.D. 2 SCCC BIO132 Lab 7 Chemical Digestion
Results: Amylase Digestion of Starch Name the enzyme: Name the substrate: Name the product: 1A 2A 3A 4A 5A 6A Amylase 10 gtt - - 10 gtt 10 gtt 10 gtt Starch - 10 gtt - 10 gtt 10 gtt 10 gtt Maltose - - 10 gtt - - - Water 10 gtt 10 gtt 10 gtt - - - HCl - - - 3 gtt - - Temperature 37 C 37 C 37 C 37 C 37 C 0 C IKI color change + or for starch Benedict s color change + or for maltose Lipase Digestion of Triglycerides Name the enzyme: Name the substrate: Name the products: 1L 2L 3L 4L 5L 4B 5B Lipase 10gtt - 10gtt (BOIL) 10gtt 10gtt 10gtt 10gtt Litmus 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt Cream - 10gtt 10gtt 10gtt 10gtt 10gtt 10gtt Water 10gtt 10gtt - - - - - Bile salts - - - - - Pinch Pinch Temperature 37 C 37 C 37 C 37 C 0 C 37 C 0 C Color change + or for fatty acids Color of tube 2L when acid was added after the experiment: Amy Warenda Czura, Ph.D. 3 SCCC BIO132 Lab 7 Chemical Digestion
Background Information Digestion = hydrolysis reactions involving enzymes (biological catalysts) -a specific enzyme acts on a specific substrate using water to break chemical bonds resulting in particular products -enzymes are usually named for their substrate and end in -ase Starch digestion by amylase (amylase) Starch (amylose) + water -------------------------> maltose Assay for enzyme (amylase) activity: Assay for starch: Lugol s IKI + starch = blue/purple/black precipitate Assay for maltose: Benedict s reagent + maltose = green, yellow, orange, red precipitate (green = less maltose, red = more) Lipid emulsification by bile (mix) Fats and oils + bile --------------------------> emulsified fats (tiny droplets suspended in water) allows easier access by water-soluble enzymes Lipid digestion by lipase (pancreatic lipase) Triglycerides + water ---------------------------------------> glycerol + fatty acids Assay for enzyme (lipase) activity: Milk cream is a source of triglycerides, litmus is a ph indicator @neutral to alkaline ph litmus is purple to blue (cream/triglycerides are neutral) @acidic ph litmus is pink (assay for fatty acids which have acid ph) Notes on Enzyme Function: Enzymes are biological catalysts that function to speed up chemical reactions by orienting molecules to favor the reaction occurring. Enzymes are specific for one type of substrate and function to catalyze only one reaction producing the same product(s) each time. Enzymes are specific for their substrate because when folded in their native conformation, enzymes have an active site that fits the particular shape of the substrate. Enzymatic reactions can be impacted by environmental conditions: -Enzymes have an optimal temperature and an optimal ph for maximum activity. -Human digestive enzymes have an optimal temperature equal to body temperature (37 C) and most have an optimal ph near neutral. -If the temperature or ph is too high, or if the ph is too low, enzymes can be denatured and will no longer catalyze the reaction. The active site will no longer fit the substrate. -If the temperature is too low, enzymes will function slowly or not at all in the reaction. Colder temperatures make molecules move slowly and thus chemical bonds are slower to react. Notes on Experimental Design: Experiments should be constructed with controls. Controls serve to insure that an experiment is working properly. Negative controls serve to show that there is no effect when there should be no effect. Positive controls serve to show the effect that is expected by a reaction occurring. By producing the anticipated results, positive controls serve as a point of comparison for the experimental results, in this case showing that expected color changes do happen with the predicted substance. Amy Warenda Czura, Ph.D. 4 SCCC BIO132 Lab 7 Chemical Digestion
Understanding the Experiment Based on the above information regarding enzyme function and the reactions being investigated, formulate hypotheses regarding the experiments and answer the following questions: 1. Tube 1A, 2A and 3A are controls. What does each control for? (Are they positive or negative controls and for what reagent? Hint: a positive control is one that shows you what the result would look like when a reaction occurs. A negative control shows you what the result looks like when a reaction did not occur.) 2. Tube 5A mimics optimal conditions (37 C, body temp). What do you predict will happen in this tube with regard to starch digestion? 3. The enzyme in tube 4A was subjected to excessively acidic ph before the incubation. What do you predict will happen in this tube with regard to starch digestion? 4. Based on the incubation temperatures, do you expect to see the same level of starch digestion in tube 6A as in tube 5A? Why or why not? 5. Tube 1L and 2L are controls. What does each control for (are they positive or negative controls)? 6. Tube 4L mimics optimal temperature conditions (37 C, body temp). What do you predict will happen in this tube with regard to lipid digestion? 7. The enzyme in tube 3L was subjected to excessively high temperature (100 C, boiling) before the incubation. What do you predict will happen in this tube with regard to lipid digestion? Amy Warenda Czura, Ph.D. 5 SCCC BIO132 Lab 7 Chemical Digestion
8. The enzyme in tube 5L was subjected to excessively low temperature (0 C, freezing) during the incubation. What do you predict will happen in this tube with regard to lipid digestion? 9. Tubes 4B and 5B were incubated at the same temperatures as tubes 4L and 5L but contained bile salts. How do you expect the digestion results in tubes 4B and 5B to compare to tubes 4L and 5L? (Think about what bile salts do to lipids and how this affects the enzyme s ability to access its substrate.) Optional Computer Activity: PhysioEx Exercise 39B (On the PhysioEx CD-ROM packaged with the lab book) pages PEx-125 to PEx-139 (back of the book) in 9 th edition pages PEx-129 to PEx-143 (back of the book) in 10 th edition PhysioEx Exercise 8 pages PEx-119 to PEx-130 (back of the book) in 11 th &12 th editions For study: Review Sheet Exercise 39A / 39 pages 605-608 in the 9 th, 10 th and 11 th editions pages 609-612 in the 12 th edition Answers in the Instructors Manual at the Eastern Campus Library on reserve Amy Warenda Czura, Ph.D. 6 SCCC BIO132 Lab 7 Chemical Digestion