Biochemistry II

Laboratory Exercise # 3

Test for effect of pH on catalysis

Title: Test for effect of pH on catalysis

Aim: To investigate the effect of pH on enzyme activity

Introduction: Enzymes(proteins) are biological catalysts that speeds up chemical reactions by

providing an alternative reaction pathway of lower activation energy, without being altered or
consumed in the reaction (unknown, n.d.). Enzymes are highly selective, in that they would
only catalyze a specific reaction. Their specificity is because of the shape of the enzyme
molecules. This part of the enzyme that has the right shape and functional group is called the
active site and the reactant that binds to those active sites is called the substrate. When the
substrate binds to the active site of the enzyme a product forms and since the enzyme reduces
the activation energy the reaction takes place faster. After the product is formed, it separates
from the enzyme which goes to catalyze another reaction. However, factors such as
temperature, substrate concentration, enzyme concentration and pH can affect enzyme activity
(unknown, n.d.).

In this lab exercise, the effect of pH on enzyme activity will be investigated. The reaction which
the researcher will be working with is the hydrolysis of starch (breaking down of starch to
simple sugars due to the reaction with water) using promalt (which contains α- amylase
activity) as the enzyme. The promalt (enzyme) and starch(substrate) concentration as well as
temperature will be held constant while the pH of the solution will vary. Iodine will be used as
starch indicator.

Materials

 Water
 Phosphate buffer solution (pH 4, pH 7, pH 10)
 2.5% starch solution
 2% Promalt solution
 Iodine solution
 Timer
 12 test tubes
 Dropper
Method

1. In one test tube 4ml of promalt solution was combined with 6ml of water and the
solution was well mixed.
2. 2ml of the diluted promalt was transferred to three test tubes, these test tubes were
labelled A-C (buffers at pH 4,7 and 10 was added to teste tubes A-C respectively).
3. Test tube B was selected and 40 drops of buffered solution (pH 7) were added
4. Into eight test tubes ,2 drops of iodine solution were added, and the test tubes were
labelled 1-8
5. 0.5ml of 2.5% starch solution was added to test tube B and the content was mixed well.
6. With the use of a dropper, 1 drop of the solution from test tube B was immediately
transferred to iodine test tube 1. The dropper was rinsed and after 1minute another drop
of the solution of test tube B was transferred to iodine test tube 2.
7. This was repeated ever 1minute interval until iodine test tube 8 was used.
8. The content of the test tubes was discarded, test tubes were thoroughly rinsed and dried
and steps 3-8 were repeated for test tube A and C

Results / Observations

Test tube (promalt Iodine test tubes Observation Inference

+starch+ buffer)

1 Dark color Presence of large

observed amount of starch

2 Dark color Presence of large

observed amount of starch
A- pH 4
3 Dark color Presence of large
observed amount of starch

4 Dark color Presence of large

observed amount of starch

5 Dark color Presence of large

observed amount of starch

6 Dark color Presence of large

observed amount of starch
 7 Dark color Presence of large
observed amount of starch

8 Dark color Presence of large

observed amount of starch

1 Light brown color no starch present

2 Light brick reed _

B- pH 7 3 Dark brick red _

4 Dark brown Small amount of

starch present

5 Dark brown Small amount of

starch present

6 Dark brown Small amount of

starch present

7 Dark brown Small amount of

starch present

8 Dark brown Small amount of

starch present

1 Dark color Presence of large

observed amount of starch

2 Dark color Presence of large

C- pH 10
observed amount of starch

3 Dark color Presence of large

observed amount of starch

4 Dark color Presence of large

observed amount of starch

5 Dark color Presence of large

observed amount of starch

6 Dark color Presence of large

observed amount of starch

7 Dark color Presence of large

observed amount of starch
 8 Dark color Presence of large
observed amount of starch

TABLE SHOWING THE COLOR CHANGE OBSERVED WHEN A DROPS OF

THE CONTENT OF THE (STARCH + ENZYME + BUFFER (AT THE
DIFFERENT pH) TEST-TUBE WAS ADDED TO THE IODINE TEST TUBES,
ALONG WITH THE INFERENCES.

Discussion: Enzymes are proteins and proteins are made up amino acids. The side chains/ side
groups on amino acids will carry charge (positively and negatively charged amino acids), these
positive and negative charge will attract contributing to the folding of enzymes (shaping the
enzymes and its active site). Changes in pH will affect the charge on amino acids consequently
there would be no ionic attraction thus the enzyme molecule will unfold changing the shape of
both the enzyme and the active site (unknown, n.d.). Enzymes may even undergo denaturation
at extreme pH. For an enzyme catalyzed reaction to occur a particular substrate must bind to
the active site of the enzyme and since enzyme are specific only when the right substrate binds
perfectly to its active site can there be a reaction between the two, but in the above scenario
where there is alteration of the shape of the enzyme active site due to changes in pH , the
substrate may no longer be able to fit the enzyme active site thus the reaction will not take
place.

The pH at which an enzyme works best (preform its job successfully is called the optimum
pH). As pH approaches closer to the optimum pH there will be an increase rate of reaction.
However, as pH surpass the optimum pH reaction rate will decrease eventually leading to
denaturation (Samantha C. 2014).

In this experiment we utilized a hydrolysis of starch reaction using promalt (which contain α
amylase activity) as the enzyme, to test for the effect of pH on enzyme activity. When
amylase reacts with starch, starch get broken down into simple sugars, knowing this we could
have assessed the activity of the enzyme at the various pH by observing the color change of
the iodine since iodine will react with starch to give a blue- black color. So, if the conditions
are favorable the amylase will break down the starch thus less and less starch will be in
solution and the iodine color should become light.
From the experiment it was observed that at pH 4 and 10 for both when the (enzyme
(promalt) + starch) solution was added to the iodine test tubes a dark color was observed (for
all 8 iodine test tubes). The reason for this is because as was explained pH can alter the shape
of the enzyme active site thus the substrate will not be able to bind for the reaction to take
place. In this case that was exactly what happened. This low pH (4) and high pH (10) altered
the shape of the active site of promlat (enzyme) thus the starch molecule (substrate) could
have not bind to it to get broken down to the simple sugars. Thus, it remained in the solution
and reacted with the iodine producing the dark color. It could have also been that the promalt
got denatured thus the starch could have not been hydrolyzed.

On the other hand, a pH 7, it was observed that when the (promalt+ starch) solution was
dropped in iodine test tube 1 no change occurred. The iodine color remained the same
indicating no starch was present in the solution. However, as time progressed (as the
promalt+ starch) solution was added to test tube 3-8) a dark brown color was observed
indicating that some amount of starch was present in the solution. This means that the pH had
some effect on the enzyme which impaired the enzyme function, thus all the starch was not
hydrolyzed. Nevertheless, based on the results obtained, when the activity of the enzyme at
the 3 different pH was compared, the enzyme worked the best at pH 7 in that at this pH some
of the starch was hydrolyzed. According to theory the optimum pH of this enzyme is
somewhere around 6.7. So, the overall trend observed complied with theory, except for the
variations in color change at pH 7 for test tubes 1-3 which may have been because of random
errors (not being able to tell the correct color change.)

Conclusion: The temperature at which an enzyme works best is known as its optimum pH
(different enzyme will have different optimum pH). pH lower and higher than that optimum
pH will cause alteration in the shape of enzymes and its active site consequently the enzyme
will not be able to carry out its function. Extreme pH can even lead to denaturation.
Nevertheless, as pH approaches the optimum pH the rate of a reaction will increase but if it
surpasses the optimum pH the reaction rate will start to decrease.
References:

 Boumis. R. (2018) “What Happens to Enzyme Activity if the pH Is Unfavorable?”

Retrieved on 6th October, 2018, Retrieved from: https://sciencing.com/happens-
enzyme-activity-ph-unfavorable-10952.html
 Ernest Z. (2014), “How does pH change protein structure?” Retrieved on 6th
October, 2018, Retrieved from: https://socratic.org/questions/how-does-ph-
change-protein-structure
 Samantha C. (2014). “How does ph affect enzyme activity?” Retrieved on 6th
October, 2018, Retrieved from: https://socratic.org/questions/how-does-ph-affect-
enzyme-activity
 BiologyWise (n.d.), “What are Enzymes and How Do They Work?” Retrieved on
6th October 2018, Retrieved from: https://biologywise.com/how-do-enzymes-
work
 Unknown, (n.d.) “Enzymes,” Retrieved on 6th October 2018, Retrieved from:
https://www.bbc.com/bitesize/guides/z89mk2p/revision/5
 BiologyWise(n.d.). “Effect of pH on Enzymes,” Retrieved on 6th October 2018,
Retrieved from: https://biologywise.com/ph-effect-on-enzymes
 Unknown ,(n.d.) “Enzymes,” Retrieved on 6th October, 2018, Retrieved from:
http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm