Happy Mariel U.

Lim February 11, 2014

BS – Chemistry III Chemistry 161: W:1:0-4:30P.M
Enzymes
Abstract
Enzymes are biological catalysts responsible for supporting almost all of the chemical
reactions that maintain homeostasis. In this experiment, enzymes highlighted are amylase and
catalase taken from saliva and potato respectively. The experiment aims to describe the role of
these enzymes in a system and also how certain environmental conditions affect its activity,
conditions such as temperature and pH changes and the presence of inhibitors. As a result, it is
found out that there are specific optimum ranges for specific enzymes to work best in a reaction.

Introduction
Enzymes speed up reactions by huge factors. And most reactions in biological systems do
not take place at perceptible rates in the absence of enzymes. And because of their roles, life
processes are maintained. Enzymes are found in all tissues and fluids of the body. Intracellular
enzymes catalyze the reactions of metabolic pathways. Plasma membrane enzymes regulate
catalysis within cells in response to extracellular signals, and enzymes of the circulatory system
are responsible for regulating the clotting of blood. Almost every significant life process is
dependent on enzyme activity.
Objectives:
1. Describe and explain the role of an enzyme in biological systems
2. Give experimental proofs on the specificity of enzymes
3. Observe and infer the effect of temperature, pH, and presence of inhibitors on the
activity of enzymes
4. Describe the optimum temperature and pH of most enzymes

Experimental Details
Part I: Activity of Enzymes
A. Amylase
1. Saliva (2mL) was collected. Made sure that the mouth was washed with water before
collecting saliva.
2. Starch solution (1%, 10mL) was obtained from the reagent area. Solution was placed in a
clean test tube. Served as sample solution for numbers 3 and 4.
3. Two test tubes were prepared.
Test tube 1: 1mL starch solution + 2 drops of iodine solution
Test tube 2: 1mL starch solution + 1mL Fehling’s A and 1mL Fehling’s B
Test tube 2 was heated in a water bath for 5 minutes while test tube 1 was set aside.
4. Remaining starch solution was mixed with the 2mL collected saliva in another clean test tube.
It was shook for a moment and immersed in a water bath maintained at 37˚C for 30 minutes.
5. After the time allotted, separate portions of the starch-saliva mixture were tested with iodine
and Fehling’s reagents. It was observed. Results were compared with the results in number 3.
B. Catalase
1. A small potato was pared and grated into fine pulp.
2. The pulp were mixed with 100mL of iced water, stand for 15 minutes and filtered through
cheesecloth.
3. Filtrate was divided into 2 parts in separate test tubes. One part was boiled in water bath.
4. Few drops of 3% H2O2 were added into each test tube. It was observed and results were
compared between the 2.

Part II: Factors Affecting the Activity of Enzymes

A. Effect of Temperature
1. Pork liver extract (1mL) was placed into 3 separate test tubes. They were immersed in a water
bath for 15 minutes:
Test tube 1: Ice bath (0˚C-5˚C)
Test tube 2: 37˚C-40˚C
Test tube 3: boiling water (95˚C-100˚C)
2. After 15 minutes, H2O2 (1mL) was added to each test tubes without removing them from the
bath.
3. The height (in cm) of the foam generated was measured after 5 minutes.
4. A graph was constructed. Height vs Temperature.
B. Effect of pH
1. Pork liver extract (1mL) was placed into 3 separate test tubes.
Test tube 1: 1M HCl (1mL) and H2O2 (1mL)
 Test tube 2: H2O2 (1mL)
Test tube 3: 1M NaOH (1mL) and H2O2 (1mL)
2. The height (in cm) of the foam generated was measured after 15 minutes.
3. A graph was constructed. Height vs pH.
C. Effect of Inhibitor
1. Pork liver extract (1mL) was placed into 3 separate test tubes.
Test tube 1: 95% ethanol (1mL)
Test tube 2: 0.1M Mercury(II) nitrate solution (1mL)
Test tube 3: distilled water (1mL)
2. The three test tubes were immersed in a water bath heated at 37˚C for 5 minutes.
3. Hydrogen peroxide (1mL) was added.
4. Height of the foam generated were compared.

Results and Discussion

Part I. Activity of Enzymes
A. Amylase

Test tube Content + Iodine + Fehlling’s A & B

1 starch Blue black solution Royal blue solution
2 Starch-saliva Yellow solution Red orange ppt; green
solution

Starch exists in 2 forms: α-amylose and amylopectin. Addition of Iodine solution to

starch results to blue black colored complex. However, α-amylose is responsible for this
coloration. Iodine inserts to the hydrophobic mid of the amylose as observed in test tube 1. While
the amylase in saliva breaks down the long chain of the starch compound into individual glucose
or smaller units. Amylase acts as an enzyme in starch hydrolysis. And that is why, negative
result is observed in test tube 2 for α-amylose is already broken down and so no iodine complex
is formed.
Fehling’s test is a general test for aldehydes and will give brick red precipitates as a
result. For test tube 1, no free aldehyde presence is detected while test tube 2, do have red brick
precipitates observed. This is due to the hydrolyzed starch that gives off glucose and other sugars
containing oxidizable functional groups like aldehydes.
B. Catalase
 Test tube Case Bubble formation
1 Heated less
2 Not heated more

Reaction:
2H2O2 2H2O + O2
The reaction above is a spontaneous reaction yet happens very slow. But with potato
enzyme – catalase, s suitable catalyst for the reaction, it is made observable very fast through the
formation of bubbles. However, temperature affects activity of enzymes. High temperature
will/may destroy the enzyme and that is why less bubble formation (foam) is observed in the
heated test tube.
Part II. Factors Affecting the Activity of Enzymes
A. Effect of Temperature

Temperature affects rate of activity of enzyme. Extreme low temperature causes less
collisions and interactions of H2O2 substrate and catalyse, thus, less decomposition is observed.
Extreme high temperature may denature the protein out of the enzyme and loses its function in
this reaction. And so for this case, catalase works best at body or room temperature (37˚C-40˚C).
Enzymes works best at specific temperature – a narrow optimum temperature range.
B. Effect of pH

According to literatures, optimum pH, where enzyme catalase works best, of the liver
extract is 7.6 as shown in the graph above. Changes in pH, high or low, can make or break intra
and intermolecular forces/ bonds, changing the shape of the enzyme, thus, changing its
effectiveness or its activity.
C. Effect of Inhibitor

Test tube Content Bubble formation

1 95% ethanol 1.00 cm
2 Hg(NO3)2 0.00 cm
3 Distilled water 2.00 cm

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and can
slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition
- competitive, non-competitive and substrate inhibition. In the results shown above, inhibitor is
the Hg(NO3)2, in which it totally stop the catalysis.

Conclusion

The activity of an enzyme is affected by its environmental conditions. Changing

these affects the rate of reaction. Environmental conditions such as temperature, pH, and
concentration of both substrate and enzyme.
 As temperature increases, initially the rate of reaction will increase, because of increased
kinetic Energy. However, the effect of bond breaking will become greater and greater, and
the rate of reaction will begin to decrease.

Small changes in pH above or below the optimum do not cause a permanent change to

the enzyme, since the bonds can be reformed. However, extreme changes in pH can cause
enzymes to denature and permanently lose their function. Enzymes in
different locations have different optimum pH values since their environmental conditions may
be different

Changing the concentration of a substance only affects the rate of reaction if it is

the limiting factor: that is, it the factor that is stopping a reaction from preceding at a higher
rate.

References

http://themedicalbiochemistrypage.org/enzyme-kinetics.php#reactions
http://www.ncbi.nlm.nih.gov/books/NBK22380/
http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146?tree=
http://www.worthington-biochem.com/introbiochem/inhibitors.html