Daiva Ahire
SCB 201
Prof. Dr. D. P. Wejesinghe
10/13/2009
Week IV
Title: Enzymes
Objectives: using spectrophometer predict the effect that the enzyme and substrate
concentrations will have on the rate of an enzyme-catalyzed reaction. Determine the effects of ph
and temperature will have on the rate of an enzyme-catalyzed reaction.
Introduction: Enzymes are made from proteins. It is a biological catalyst that speeds up the rate
of chemical reaction by lowering the activation energy of the reaction. Enzymes are specific and
catalyze only specific reaction. Specificity is a result of active site of enzyme that acts on the
substrate. The enzyme activity is influenced by ph and temperature. Change in ph will have
impact on hydrogen bonding that can change the shape of protein. Increase in the temperature
will increase the reactions rate; however, if the temperature is too high the enzymes will
denatures. For this experiment we use spectrophometer which measures the absorbance of the
solution for particular wavelength.
Experiment I- prediction of the effect of the enzyme concentration to the rate of reaction.
Hypothesis: Higher concentration of the enzyme will increase the rate of the reaction; therefore
the absorbance also will increase.
Procedure: first we calibrate the spectrophometer with blank solution. We measured 1ml of
Catecholase in the test tube using 1 ml graduate pipette and added 5 ml of distilled water.
Covered the test tube with Parafilm and mixed the content. On the spectrophometer we set the
wavelength to 540 nm, because this wavelength is best absorbed by the solution. Using the left
knob on the machine we set absorbance reading to infinity. We inserted the test tube into sample
holder. Using the knob on the right we set the absorbance reading to 0. In the mean time we
prepared three test tubes in each added 2 ml of substrate Catechol with 3 ml, 3.5 ml, and 4 ml of
water respectively. The last is added enzyme, because the reaction will take place as soon as we
add the enzyme. The amount of the substrate Catechol is constant. We are changing the
concentration of the enzyme Catecholase ( 1ml, 0.5ml, 0 ml) by diluting it with water. Therefore,
the concentration of enzyme is independent variable. The dependent variable is absorbance,
which will be changing according to concentration. We mixed the contents of the test tubes.
After 3 min. we used blank to rezero the spectrophometeer, inserted the first test tube and noted
the absorbance. Same procedure we conducted for the rest of the test tubes.
Results:
Tube
Water (ml)
3
Catechol (ml) substrate
2
Catecholase
(ml)- enzyme
1
1
2
3.5
2
0.5
3
4
2
0
Absorbance and
color intensity
0.9 – intensive
brown color.
0.6 – brownish
color
0 – off white
color
Daiva Ahire
SCB 201
Prof. Dr. D. P. Wejesinghe
10/13/2009
Week IV
Conclusions: The results of the experiment showed that lowering the concentration of enzyme
and adding more water, the absorbance of the solution decreases. Therefore, our experiment
supports our hypothesis that higher concentration of enzyme will increase the rate of reaction.
Higher concentration of enzyme - higher absorbance and color intensity. Graphic relationship:
Experiment II – effect of pH to the enzyme activity.
Hypothesis: different concentration of hydrogen ions, therefore different ph, will have some
effect on the enzyme activity and the reaction rate. The normal ph for living organisms cells is
neutral (about 7), therefore we expect that the absorbance and the highest rate of the reaction
should occur at the neutral ph.
Procedure: We prepared three test tubes by adding constant amount of 2 ml of substrate
Catechol, 3ml of different ph solution (pH 4, pH 7 and pH 9) and 1ml of enzyme Catecholase.
Independent variable in this experiment is ph value and dependent variable is absorbamce.We
mixed the contents of the test tubes. After 3 min. we used blank to rezero the spectrophometeer,
inserted the first test tube and noted the absorbance. Same procedure we conducted for the rest of
the test tubes.
Results:
Test tube
Ph solution and
Catechol (ml) Catecholase
Absorbance and
amount taken (ml)
substrate
(ml)- enzyme
color intensity
1
pH 4 - 3
2
1
0.4
2
pH 7 - 3
2
1
1.3 –intensive
brown color
3
pH 9 - 3
2
1
0.3
Conclusions: The results of the experiment showed that the optimal enzyme activity occurs in
ph 7 solution. In supports our prediction that most enzyme activity occur in the neutral solution.
The alkaline solution with pH 4 showed higher absorbance compared with acidic solution with
pH 9. Graphic illustration of the relationship ph vs. absorbance:
Daiva Ahire
SCB 201
Prof. Dr. D. P. Wejesinghe
10/13/2009
Week IV
Experiment III – effect of the temperature to the enzyme activity.
Hypothesis: Since the increase of the temperature will increase the number of collisions between
enzyme and substrate we expect the increase in the rate of reaction. However, if the temperature
will be too high, we expect that enzyme will change its structure, perhaps even be denatured and
enzyme will be not able to catalyze the reaction.
Procedure: We took first test tube and determined the absorbance at about 0 C (ice). We added
3 ml of water and 2 ml of substrate and kept in the ice bucket. As the solution in the test tube
cool down we added 1 ml of enzyme. Mixed the content of the solution, left the test tube for the
rest 3 min. in the ice bucket and then checked the absorbance. The second test tube we prepared
in room temperature. We added 3ml of water, 2 ml of substrate and 1 ml of enzyme, all
substances were at room temperature. Mixed the content of the solution, left the test tube for 3
min. at room temperature and then checked the absorbance. The third test tube we prepared at
high temperature. We took 3 ml of water and 2 ml of substrate and placed in the hot water bath.
After the temperature of solution increased we added 1ml of enzyme solution, which also was
kept at the high temperature. We mixed the content of the solution, left the test tube for 3 min. in
the water bath and then checked the absorbance. In this experiment temperature is independent
variable and absorbance is a dependent variable.
Results:
Test tube
condition
Water (ml)
Catechol (ml) Catecholase
Absorbance
-substrate
(ml)- enzyme
and color
intensity
1
Ice (0 C)
3
2
1
1.0
2
Room
3
2
1
0.9
temperature
(24 C)
3
Hot water
3
2
1
0.6
bath (40 C)
Daiva Ahire
SCB 201
Prof. Dr. D. P. Wejesinghe
10/13/2009
Week IV
Conclusions: The results of the experiment showed that ice and room temperature substances
had the highest absorbance. Test tube kept in the ice bucket showed the highest absorbance.
Therefore, our experiment result contradicts our hypothesis. At very high and very low
temperatures we expected the absorbance or enzyme activity be low. Te highest absorbance
should have appeared at room temperature, because most human enzyme activity occurs at room
temperature. I believe that mistake occur in preparing the test tube in ice conditions. The
absorbance result is very close to room temperature test tube, which indicates, that we probably
didn’t cool the solution very well. Therefore, after adding enzymes the reaction proceeded as if it
is in room temperature. The test tube prepared in the hot water bath also showed some of the
enzyme activity. I suggest that next time conducting this experiment it is useful to use
thermometer in order to check the accuracy of the temperatures. Graphic representation of the
relationship between temperature and absorbance of this experiment:
Homework:
1. A freshly cut potato turns brown when left standing. Why do mashed potatoes stay
white?
Mashed potatoes are cooked. The heat from cooking them denatures the enzyme Catecholase
which causes raw potatoes to turn brown through oxidation when they are exposed to air.
2. The three graphs in Figure 11 represent three different enzymes that are found in a
unicellular pond organism. Assuming that these enzymes must function in order for the
organism to grow, what water pH is best for this organism? Explain your answer.
Water pH 5 is the best for this organism to survive. In all three graphs the highest activity of the
enzyme is at the pH 5 - the highest point of the curve.
3. Lemon juice, which has a pH of about 3, can be sprinkled on freshly cut fruit to keep it
from turning brown. Propose a hypothesis to explain this observation.
Daiva Ahire
SCB 201
Prof. Dr. D. P. Wejesinghe
10/13/2009
Week IV
The lemon juice with pH3 will denatures the polyphenol oxidaze enzyme presented in most of the
fruits. Therefore the fruit will not turn brow.
How could you test your hypothesis?
We can cut one fruit and leave it open, which will expose enzyme to oxygen and the fruit will turn
brown fast. On the other fruit we can sprinkle the lemon juice and observe the changes.
Propose an alternative hypothesis. (Hint: Lemon juice is a complex substance.)
4. You hypothesize that reaction rate is proportional to enzyme concentration and design
an experiment to test your hypothesis. Fill in the blanks below to complete the design.
Test tube
Tube A
Tube B
Tube D
Explain your answers:
Water
20 drops
15 drops
10 drops
0 drops
Substrate
10 drops
10 drops
10 drops
10 drops
Enzyme
0 drops
5 drops
10 drops
20 drops
Water: Water is used to dilute the concentration of the enzyme; therefore we minimize the
amount of water to 0 in the test tube D, where the concentration of enzyme is the highest.
Substrate: Substrate concentration is constant.
Enzyme: Enzyme concentration we decreased from 20 drops to 0 drops, because it is our
independent variable and we measured how it’s concentration will affect the rate of the reaction.
5. A student team is studying the effect of substrate concentration on the rate of an
enzymatic reaction. They supply each reaction mixture with the same amount of enzyme.
Predict what the results will be and explain why.
The rate of reaction will be the same, because the students didn’t change the enzyme
concentration. The enzyme concentration is directly proportional to reaction rate. If there is no
increase in concentration, the reaction rate will remain the same.