Chemical and Physical Processes of Digestion

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Chemical and Physical Processes of Digestion
Exercise 39A / 39 (begins page 435 in 8th edition, page 597 in 9th and 10th editions, page 595 in 11th ed.)
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 (37C, 0C) and boiling (100C) 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       37C        37C        37C          37C        37C         0C

        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 100C
           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     37C      37C      37C            37C       0C       37C       0C

        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 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

                                 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                  37C             37C         37C             37C          37C           0C
   IKI color change


   Starch + or 


   Benedict's color
   change

   Maltose + or 




Lipase Digestion of Triglycerides

                             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               37C        37C      37C                37C       0C         37C         0C
   Color change


   Fatty acids + or 



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:
               Litmus cream = milk cream (triglycerides) + litmus 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.
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 (37C) 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 producing the anticipated results,
positive controls serve as a point of comparison for the experimental results.



   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 the experiment works. A negative control shows you what the result looks like
   when a reaction did not occur.)




2. Tube 5A mimics optimal conditions (37C, 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 (37C, 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 (100C, 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
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