Lab No.

2
(Catchy Title)

OBJECTIVES

1. To isolate and determine the properties of catalase.

2. To determine the effect of sulfides on catalase activity.
3. To prove the presence of a protein factor in enzymes.
4. To determine the effect of temperature on enzyme activity.

MATERIALS

● 1 pc. small potato with peel ● guaiac solution

● filter paper ● saliva
● 6% H2O2 ● starch solution
● filter paper or cotton/gauze ● acidified iodine solution
● 0.1 N NaCl ● blender
● 0.05 N HCl ● funnel
● 0.05 N NaOH ● cheesecloth
● wooden splint

PROCEDURES

I. A. Preparation of Catalase Enzyme

Grate a potato, including the peel, into a fine pulp (a blender may be used for this). Add
50 mL of water in a beaker and allow the mixture to stand for 10 minutes, swirling
occasionally. Strain through cheesecloth and discard the residue. Filter again through
filter paper, (NOTE: If filtering through filter paper takes too long, use cotton or gauze.
Use the filtrater the following tests:

Mix 5 mL of the filtrate and 5 ml of 6% H2O2 in a test tube. Immediately insert a glowing
wooden splint. What gas is indicated present?

Observation: Small bubbles formed at the top of the mixture and the starch settled down
at the bottom.

B. The Protein Nature of Enzymes

 To 5 mL of the catalase extract, add 5 mL of 3 M NaOH. Mix the two solutions by
tapping the tube against the palm of your hand. Add five drops of CuSO4 solution to the
mixture. Observe and record the color.

Observations:
Potato + H2O + NaHO
- The solution cleared up; the starch that was settled at the bottom of the tube
spread around making the solution foggy.
Potato + H2O + NaHO + CuSO4
- The CuSO4 turned into a blue cloudlike form at the bottom of the test tube; brown
cloudy (probably the potato juice) and jelly-like material formed on top of CuSO4.
- The clear solution turned back into it’s tea-like color.

II. A. Preparation of Enzyme

Extract 10 to 15 ml. of saliva from a student donor and use it to test for the effects of pH
and temperature.

B. Effect of pH

Label three test tubes and mix the substances indicated below.

Tube number 1% Starch 0.1 M NaCl Acid/Base/Wat Saliva

Solution er Added

1 10 mL 1 mL 1 mL 0.05 N 2 mL
HCl

2 10 mL 1 mL 1 mL distilled 2 mL
water

3 10 mL 1 mL 1 mL 0.05 N 2 mL
NaOH

Mix well by shaking each test tube and place in a water bath maintained at body
temperature (37°CI). Recording the time at 3-minute intervals, test for the presence of
starch using 0.001 N iodine solution. Record the time needed for the blue color of starch
with iodine to fail to appear, This means that starch has been completely hydrolyzed to
glucose.

Color of Saliva Extract With Iodine

 Time (minutes) Test Tube 1 Test Tube 2 Test Tube 3

3 Purple Purple (small hints) Clear

6 Purple Purple + Brown Clear

(Iodine)

9 Purple Purple + Brown Clear

(Iodine)

12 Purple + Brown Purple + Brown Clear

(Iodine) (Iodine)

15 Purple Purple Clear

18 Purple Purple + Yellow Clear

(Iodine)

pH pH 3 Not Tested pH 12

In what test tube is there evidence of unhydrolyzed starch after 18 minutes?

Observations:
Test Tube 1
- Cornstarch settled at the bottom of the tube; the saliva turned into a stringy
texture.
- The top is clear.
Test Tube 2
- Cornstarch settled at the bottom.
- The top is mildly foggy.
Test Tube 3
- Cornstarch settled at the bottom.
- The top is foggy white.

What is the optimum pH of salivary amylase?

- The optimum pH of amylase is between 6.7 and 7.0, which is close to neutral

Conclusion:

C. Effect of Temperature

Label three test tubes, add 10 mL of 1% starch suspension and 1 mL 0.1 N NaCl solution.
Immerse tube 1 in a beaker of water at room temperature, tube 2 in a water bath at 37°C;
and tube 3 in a boiling water bath. Using a pipette, add exactly 2 mL of saliva into each
tube. Mix well and monitor in a manner similar to Part II-B. Note the length of time it
takes before the starch is completely hydrolysed. Record the time needed before the blue
color with iodine no longer appeaгs.

Color of Saliva Extract With Iodine

Time (minutes)
Test Tube 1 Test Tube 2 Test Tube 3
(Room (Water at 37° C) (Boiling Hot Water)
temperature)

3 Brown (Iodine) Purple (after 1 Brown (Iodine)

minute)

6 Blue upon dropping Iodine + white Purple + Iodine

but gradually particles were
disappeared floating

9 Brown (Iodine on Iodine (faded 1 and Purple

top) a half minute)

12 Brown (Iodine on Iodine (faded 1 and Blue-violet

top) a half minute)

15 Brown (Iodine on Iodine (faded 1 and Purple

top) a half minute)

18 Brown (Iodine on Iodine (faded 1 and Purple

top) a half minute)

Observations: ● Iodine ● Iodine ● Iodine

settled at the settled on turned
top while top while purple on
the iodine the starch top.
settled at the settled at the ● Starch
bottom. bottom. settled at the
● Small hints ● Iodine faded bottom in an
of purple overtime. opaque and
floated at jelly-like
the top, in texture.
the very
center of the
iodine.

QUESTIONS:
 1. Write the equation for the reaction catalyzed by catalase.
- 2H2O2 => 2H2O2 + O2
2. How does an enzyme alter the rate of chemical reaction?
- Enzymes (and other catalysts) act by reducing the activation energy,
thereby increasing the rate of reaction.
3. What is the optimum pH and temperature for salivary amylase?
- In vitro activity of human salivary alpha-amylase showed the optimum pH
and temperature at 7.0 and 37 degrees C, respectively.
4. What effects do increased pH and increased temperature have on the activity of
salivary amylase?
- At higher temperature the enzymes are denatured, while at lower
temperature, the enzymes are deactivated, so this takes more time at low
and high temperature to digest the starch. At optimum temperature (32–37
°C), the enzyme is active and therefore consumes less time for starch
digestion.
5. In summary, what factors affect enzyme activity?
- Enzyme activity can be affected by a variety of factors, such as
temperature, pH, and concentration.

CONCLUSION/GENERALIZATION