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: Starch 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 PREPARATIONS1
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
13 100 mm (Carolina 73-0008)
Standard
16 150 mm (Carolina 73-0014)
Medium large
20 150 mm (Carolina 73-0019)
Large
25 150 mm (Carolina 73-0025)
1
1 cm = 1.0 ml
1 cm = 1.5 ml
1 cm = 2.4 ml
1 cm = 4.0 ml
Note: “Materials and Preparations” instructions are grouped by exercise. Some materials may be used in more than one exercise.
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7.1 Starch Digestion by Salivary Amylase (LM pages 92-93)
_____
amylase solution (Carolina 20-2350)
_____
boiling water bath
_____ test-tube holder
_____ hot plate
_____ beaker
_____ beaker tongs
_____
starch solution (Carolina 89-2530)
_____
iodine-potassium-iodide (IKI) solution, premade (Carolina 86-9051, -9053, -9055)
_____
Benedict’s reagent powder (Carolina 84-7091) or Benedict’s reagent solution (Carolina
84-7111, -7113)
Amylase solution. Prepare 20 ml per student group. Dissolve 1 g alpha amylase in 100 ml distilled water.
Starch 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 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.
7.2 Fat Digestion by Pancreatic Lipase (LM pages 94-95)
_____
pancreatin (pancreatic lipase) (Carolina 87-8928)
_____
vegetable oil, preferably canola, olive, or sunflower
_____
phenol red solution (Carolina 87-9850 to 87-9854)
_____
incubator, 37°C
_____
bile salts (oxgall, Wards Biology 38W 2179). One gram is enough for a class.
Pancreatin solution (1% pancreatin in 0.1% Na2CO3). Prepare 30 ml per student group. Add 1 g pancreatin
to every 100 ml of 0.1% Na2CO3 (0.1g Na2CO3 per 100 ml of distilled water).
Phenol red 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 Pepsin (LM pages 95-96)
_____
1–2% pepsin solution (Carolina 87-9378, -9380)
_____
incubator, 37° C
_____
albumin solution (Carolina 84-2250, -2252)
_____
0.2% hydrochloric acid (HCl) (Carolina 86-7790,-7791)
_____
biuret reagent (Carolina 84-8213)
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 84-9380) 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.
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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.
EXERCISE QUESTIONS
7.1 Starch Digestion by Salivary Amylase (LM pages 92-93)
1. Why is this reaction called a hydrolytic reaction? Starch 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 Starch 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: Starch Digestion (LM page 92)
Table 7.1 Starch Digestion by Amylase*
Tube
Contents
1
Alpha-amylase
Starch
Time
0
Type
of Test
Results
Explanation
Starch (iodine)
Positive: black
No digestion; not enough time
Sugar (Benedict’s) Negative: blue
No digestion; not enough time
Digestion; enough time elapsed
2
Alpha-amylase
Starch
0
3
Alpha-amylase
Starch
30
Starch (iodine)
4
Alpha-amylase
Starch
30
Sugar (Benedict’s) Positive:
green-orange
Digestion; enough time elapsed
5a
Alpha-amylase,
boiled
Starch
30
Sugar (Benedict’s) Negative: blue
No digestion; enzyme is denatured
5b
Alpha-amylase,
boiled
Starch
30
Starch (iodine)
No digestion; enzyme is denatured
6
Water
Starch
30
Sugar (Benedict’s) Negative: blue
Negative:
dark purple
Positive: black
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.
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7.2 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
Initial
Final
Explanation
1
Vegetable oil
Phenol red
Bile salts
*
Red
Red
No digestion (no enzyme); control
2
Vegetable oil
Phenol red
Pancreatin
*
Red
Pink (red
at border)
Limited digestion; no emulsifier
3
Vegetable oil
Phenol red
Pancreatin
Bile salts
*
Red
Yellow
Digestion; enzyme and emulsifier present
4
Vegetable oil
Phenol red
Distilled water
*
Red
Red
No digestion (no enzyme or emulsifier);
control
*
*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? Phenol red 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 Pepsin (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 Pepsin
Tube
Contents
Temperature
Results
of Test
Explanation
1
Distilled water
Albumin
37°C
Purple
No digestion (no enzyme); control
2
Albumin
Pepsin
Distilled water
37°C
Purple
No digestion; incorrect pH
3
Albumin
Pepsin
HCl
37°C
Pinkish-purple
Digestion; enzyme and correct pH
4
Albumin
Pepsin
HCl
22°C
Light purple
to pale
pinkish-purple
Some digestion; temperature is low
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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
Explanation
Specific enzyme
Each enzyme speeds only one type of reaction.
Specific substrate
Enzyme and substrate must come together like a lock and key for reaction to occur.
Warm temperature
Chemical reactions occur at a faster rate at warm temperatures than at cold temperatures.
Time
It takes time for the enzyme to bring about the reaction.
Specific pH
Optimum pH maintains the shape of the enzyme so that the enzyme will combine with
its substrate.
Fat emulsifier
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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
When iodine (IKI) solution turns blue-black, what substance is present? starch
What color is Benedict’s reagent originally? blue
What happens to an enzyme when it is boiled? denatured
Saliva contains what enzyme? amylase
As oil is digested, why does the first tube turn from red to yellow? Fatty acids are released.
What temperature promotes enzymatic action? body temperature
What do you call a sample that goes through all the steps of an experiment, but lacks the factor
being tested? control
What role do bile salts play in digestion of fat? an emulsifier
What color does biuret reagent turn when peptides are present? pinkish-purple
Is the optimal pH for pepsin acidic or basic? acidic
Why would you predict that pepsin would not digest starch? Enzymes are specific.
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. Pepsin, protein, water, body temperature
b. Pepsin, 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.