Experiment 2

Introduction
On this experiment, we are focusing on enzymes. Enzymes are biological catalysts (also
known as biocatalysts) that speed up biochemical reactions in living organisms, and which
can be extracted from cells and then used to catalyse a wide range of commercially important
processes. Enzymes work as lowering the activation energy and needed to make a chemical
reaction occur. Similar to other catalysts, enzymes change the equilibrium of the reaction but
they are not consumed in the process and the enzymes are very specific. Most enzymes are
globular protein that are larger than substrate which they are interact with. Enzymes also have
their active sites which contain one or more binding sites that specific to the substrates in
correct configuration.

Activity 1: Calibration and Settings for Spectrophotometer

Materials
1. Spectrophotometer
2. Calibration cuvette
3. Starch in dropper water
4. Distilled water
5. Parafilm squares

Procedure
1. The spectrophotometer turned on and let it warmed up for 5 minutes.
2. The calibration cuvette prepared:
1) The cuvette was marked at the 2 cm level.
2) Distilled water added until the 2 cm level.
3) 10 drops of starch added. The parafilm was placed over the opening of the cuvette
and mixed the contents by inverting the tube several times.
3. The spectrophotometer was set at 540 nm.
4. The spectrophotometer was set to Absorbance (A)
5. The chamber cover opened and the calibration was inserted into the holder.
6. The cover closed and read Absorbance and recorded.

Activity 2: The Effect of Enzyme Concentration on the Rate of Reaction

Materials
1. Spectrophotometer
2. 4 cuvettes
3. Starch in dropper water
 4. Enzyme (amylase) mixture with cover (potato juice/ distilled water)
5. Dropper (for enzyme mixture)
6. Distilled water (bottle)
7. Parafilm squares
8. Marking pen
9. Plastic ruler
10. Spreadsheet
11. Data Collection Sheet

Procedure
1. 4 cuvettes were labelled on top of the cuvettes.
2. By using plastic ruler, 2 cm was measured from the bottom of each cuvettes.
3. Each cuvettes were filled by distilled water to the 2 cm mark.
4. Preparations of cuvette contents:
1) 5 drops of enzymes added to cuvette 2, 15 drops to cuvette 3 and 45 drops to cuvette
4 were added.
2) Added up 10 drops of starch to each of the tubes.
3) The cuvettes were set for 5 minutes.
5. Cuvette 1 at the end of 5 minutes was placed in the spectrophotometer and measured its
absorbance.
6. Step 5 was repeated with cuvettes 2, 3 and 4.
7. The results recorded.
8. The cuvette’s contents emptied into waste container provided.
9. The cuvettes were rinsed out and let them dried.

Activity 3: The Effect of Substrate Concentration on the Rate of Reaction

Materials
1. Spectrophotometer
2. 7 cuvettes
3. Starch in dropper water
4. Enzyme (amylase) mixture with cover (potato juice/ distilled water)
5. Dropper (for enzyme mixture)
6. Distilled water (bottle)
7. Parafilm squares
8. Marking pen
9. Plastic ruler
10. Spreadsheet
11. Data Collection Sheet

Procedure
1. Seven cuvettes were numbered.
 2. Using the plastic ruler, 2 cm from the bottom of cuvettes were measured and a mark
was marked with the marking pen.
3. Each the cuvettes was filled with distilled water to the 2 cm mark.
4. Preparation of cuvette contents:
1) 1 drop of of starch added to cuvette 1, 3 drops for cuvette 2, 5 drops to cuvette 3, 10
drops for cuvette 4, 20 drops to cuvette 5, 40 drops to cuvette 6 and 60 drops to
cuvette 7.
2) 10 drops of amylase added to each of the 7 cuvettes.
3) The cuvettes were set for 10 minutes.
5. At the end of 10 minutes, cuvette 1 was placed in the spectrophotometer and measured
its absorbance.
6. Step 5 was repeated with cuvettes 2, 3, 4, 5, 6 and 7.
7. The results were recorded.
8. The cuvette’s contents emptied into waste container provided.
9. The cuvettes were rinsed out and dried them.

Activity 4: The Effect of pH on Enzyme Activity

Materials
1. Spectrophotometer
2. 6 cuvettes
3. Starch in dropper water
4. pH Buffers 3, 5,
5. Dropper (for enzyme mixture)
6. Distilled water (bottle)
7. Parafilm squares
8. Marking pen
9. Plastic ruler
10. Spreadsheet

Procedure
1. Six cuvettes numbered.
2. Using the plastic ruler, 2 cm from the bottom of cuvettes were measured and a mark
was marked with the marking pen.
3. Preparation of cuvette contents:
1) Cuvette 1 filled up to the 2 cm with pH 3 buffer and 10 drops of enzyme.
2) Cuvette 2 filled up to the 2 cm with pH 5 buffer and 10 drops of enzyme.
3) Cuvette 3 filled up to the 2 cm with pH 7 buffer and 10 drops of enzyme.
4) Cuvette 4 filled up to the 2 cm with pH 9 buffer and 10 drops of enzyme.
5) Cuvette 5 filled up to the 2 cm with pH 11 buffer and 10 drops of enzyme.
6) Cuvette 6 filled up to the 2 cm with distilled water and 10 drops of enzyme.
7) Let the cuvettes stood for 5 minutes.
4. At the end of 5 minutes, cuvette 1 was placed in the spectrophotometer and measured
its absorbance.
5. Step 5 was repeated with cuvettes 2, 3, 4, 5, 6 and 7.
6. The results were recorded.
 7. The cuvette’s contents emptied into waste container provided.
8. The cuvettes were rinsed out and dried them.

Results
Lab Activity 1

CUVETTE OD
1 0.053

Lab Activity 2

CUVETTE OD
1 0.629
2 0.119
3 0.057
4 0.063

The effect of Enzyme Concentration on the Rate of Reaction

0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40 45 50
Enzyme Concentration

Lab Activity 3

CUVETTE OD
1 0.044
2 0.048
3 0.054
4 0.053
5 0.055
6 0.056
7 0.053
 The effect of Substrate Concentration on the Rate of Reaction
0.06
0.05
0.04
0.03
OD

OD
0.02
0.01
0
0 10 20 30 40 50 60 70
Substrate Concentration

Lab Activity 4

CUVETTE OD
1 0.052
2 0.057
3 0.050
4 0.043
5 0.046
6 0.053

The effect of pH on Enzyme Activity

OD
0.02
0.01
0
3 5 7 9 11 7
pH Concentration
Discussion
Various environmental factors contributing to the effect of the rate of enzyme reactions.
Based on this experiment, we were doing the effect of enzyme concentration, substrate
concentration and pH on rate of reaction.
The rate of reaction involving enzymes increase as the concentration increase. The original
graph for enzyme concentration is in linear because when the concentration of enzyme
increase, the reaction also become faster as the substrate is constant concentration. Enzyme
concentration is directly proportional to the rate of reaction. Thus, we will get simple linear
graph relationship between the reaction rate and the enzyme concentration.
The situation above is similar on the substrate concentration. If the substrate concentration
increase, the rate of reaction also increase. As the concentration of the substrate increase, the
sites are bounded greater until the saturated. When the enzyme saturated, the binding site are
not available for the substrate and the rate of reaction will be slow.
The optimum pH for the enzymatic activity of salivary amylase ranges from 6 to 7. Above
and below this range, the reaction rate reduces as enzymes get denaturated. The enzyme
salivary amylase is most active at pH 6.8. The active sites on enzymes are often composed of
ionizable groups which must be in the appropriate ionic form to maintain the conformation of
the active site, bind the substrates or catalyze the reaction. On the other hand one or more of
the substrates may contain ionizable groups and only one ionic form of that substrate may
bind to the enzyme or undergo catalysis.
The result I got for each experiment may different because some mistakes during the
experiments. For example, not used the spectrophotometer properly or maybe misplacing the
cuvettes in the spectrophotometer. Otherwise, mistake from doing the dropping process of
enzyme, substrate or pH concentration.

Conclusion
From the experiment, we are able to learn the factors that effect the rate on reaction such as
enzyme concentration, substrate concentration and also pH. We are also know the optimum
rate of reaction from each factor such as pH as pH 7 is optimum for enzyme amylase. We
also able to plot the graphs for each factor that contributing the effect of rate of reaction.

References

 Suckling K.E. (1984) The impact of enzymology in biochemistry and beyond. In:
Suckling C.J. (eds) Enzyme Chemistry. Springer, Dordrecht
 http://amrita.olabs.edu.in/?sub=79&brch=18&sim=236&cnt=1
 http://essays.biochemistry.org/content/59/1