Effect of different pH on Enzyme Activity. EEI: 2011- Year 11 Biology

Written by: Donnet, Luke

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Contents Introduction: ...................... 3

Aim: ................................... 4

Hypothesis: ........................ 4

Intro: .................................. 4

Materials: ........................... 4

Methods: ............................ 5

Results: .............................. 6

Discussion: ........................ 8

Conclusion: ...................... 11

Bibliography: ................... 12

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Introduction: Enzymes are protein molecules which are biological catalysts which

breakdown organic matter and lower the activation energy that is required to process the

compound. The body uses catalysts to unlock the energy that is stored within the organic

compounds it breaks down. There are three types of catalysts that are primarily utilized in the human body which are; amylase, protease and lipase. Each enzyme is specialized which

means that on them breaks down one specific type of compound. Amylase is an enzyme that

breaks down starches and into simple sugars. Amylase can be found in human saliva, this is why bread can taste sweet when you leave it on your tongue for a while because the starch is

breaking down into a simple sugar. Amylase is created in the human pancreas, salivary glands

and in the small intestines. Protease is responsible for breaking down protein compounds which leave amino acids (the building blocks of proteins). Lipase is the last enzyme that is

used in the human body and it is responsible for the breaking down of fats and oils.

(Wikipedia: Enzymes, 2011)

The primary reason why the body needs to breakdown complex compounds is for transportation. For example, protease breaks down protein compounds into amino acids. Once

these compounds are at their simplest form, they are then transported to where they are

needed (muscles). Once there, they are then re-synthesised back into proteins which are used to build muscles. Another reason why we need enzymes is because there are some compounds

which the human body process too slowly or not at all. Enzymes speed this process of

breakdown complex compounds for the body to utilize. Starch itself is not used as energy in the body; instead it needs to be broken down into a simpler compound, sugar. Starch, being a

polysaccharide (meaning: a carbohydrate which is made up by a whole bunch of

simpler/small carbohydrates) can be easily broken down into glucose which is a

monosaccharide (meaning: a simple carbohydrate). Glucose itself can and is used in the human body as energy. (Beta Force: What are Enzymes and Why We Need Them, 2006)

Each enzyme is specialized which means it can only break down one particular type of compound. Amylase

is an enzyme that breaks down starch molecules only,

which means that no other enzyme can break down starch and Amylase cannot breakdown any other

compound. This happens because the active site of an

enzyme can only support one type of substrate. (For

example, amylase can only have starch as it’s substrate). This is because the active site of an amylase cannot

support any other type of substrate other than starch

molecules. A simple way to explain this method is by the key and lock concept. The key is the substrate and

the lock is the active site, only one key will fit the lock.

(How Stuff Works: How Cells Work – Enzymes at Work, 2011)

There are three main parts to an enzyme reaction which occurs every time a complex

compound is broken down; the enzyme itself, active site and the substrate. The active site of an enzyme is basically where the complex molecules are broken down into simpler

compounds. The substrate is the molecule that is about to be broken down by the enzyme’s

active site.(Biology: A Contextual Approach, 2004)

Diagram. 1

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There are times when enzymes don’t work like they are supposed to and when this happens,

compounds called coenzymes assist the active site in breaking down its intended substrate. This happens because the active site changes its shape so the substrate is unable to properly

mount onto the enzyme. It is proven that changes in the environment (such as pH and

temperature) can alter the shape of the active site where it can no longer be used. When this happens, the enzyme is rendered as “denatured”. (Biology: A Contextual Approach, 2004)

Iodine solution is a colour indicator which detects starch molecules. The iodine indicator is a

yellowish/gold which turns into a dark purple colour upon contact with starch molecules.

Iodine is a heavy element which is denser than many other liquids.

(lugolsiodinesolution.com: Lugol's iodine, 2007)

Previous studies have found that extremes in the pH can alter the physical/chemical properties

of starch. The result of this is modified starch which is commonly known as the “E” series. (Wikipedia : Modified Starch, 2011)

Aim: The aim of this investigation was to determine the effect on amylase activity in a

range of different pH environments.

Hypothesis: Previous studies have shown that enzymes denature more

frequently towards the extreme ends of the pH scale. Therefore the rate at which the

reaction occurs will be slowed down due to the increased amount of denatured

enzymes in the more extreme environments.

Intro: In this experiment, amylase solutions have been tested in confined, acidic

and basic environments, this done to simulate the enzyme activity in the human body,

which can give us additional knowledge of their purpose in the human body. The data

collected from this experiment could prove useful for diagnosis of patients who might

be suffering from enzyme deficiencies. In this experiment, the independent variables

will be the changes in the pH at which the reaction occurs and the dependent variable

will be the colour changes during the pH test. The amount flour put into the test tubes

are the independent variables, the colour and turbidity changes are the dependent

variables in the pre-tests. The volume of the solution, temperature, amount of amylase

solution added and the shape of the test tubes are kept as constant as possible.

Materials: Material/Equipment Justification

9 test tubes A place for all the reactions

occur

100ml of 2% amylase solution It’s the enzyme used to

conduct this experiment

500g of flour A source of starch for the

enzyme to react with

300ml of 8mol hydrochloric

acid

The strong acid used to

conduct experiment

300ml of 2mol hydrochloric A weaker acid used in this

Diagram. 2

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acid experiment.

500ml of water The neutral/controlled that was

used in the experiment.

300ml of 8mol of sodium

hydroxide

The strong base chemical that

is used in the experiment

300ml of 2mol of sodium

hydroxide

The weaker base that is used

in the experiment

Iodine solution A starch colour indicator

1ml liquid dropper Used to accurately drop the

same amount of solution in

every time

Spatula Used to put power in the test

tube

Safety goggles Protect eyes from chemicals

Test tube rack Hold the test tubes through the duration of the experiment

Beaker Used as a safe way to decant

liquid from the bottles into the

test tubes.

Methods:

Conducting Pre-tests

Step 1. Put four test tubes in a single file on the test tube rack

Step 2. From left to right, mark each of the test tubes as follows; “0.5”, “1”, “1.5”, “2”.

Arrange them in ascending order on the test tube rack.

Step 3. Put the amount of flour that is displayed on the test tube from Step 2. (ie, the

test tube marked “0.5” should have 0.5 grams of flour inside it)

Step 4. Put 10ml of water into each test tube using a measuring cylinder.

Step 5. Squirt 1ml of 2% amylase solution using a pipette.

Step 6. Put 5 drops of iodine solution into each test tube.

Step 7. Shake the 4 test tubes and record any colour changes. (results best presented in

a table)

Conducting Test

Step 1. Put five test tubes in a single file in the test tube rack.

Step 2. Label the test tubes from left to right (S 8m, S 2m, N, H 2m, H 8m)

Step 3. Using the spatula and electronic scales, accurately measure 0.5 grams of flour

into each test tube.

Step 4. Put;

5ml of 8mol Sodium Hydroxide into the test tube marked “S 8m”

5ml of 2mol Sodium Hydroxide into the test tube marked “S 2m”

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5ml of water into the test tube marked “N”

5ml of 2mol Hydrochloric Acid into the test tube marked “H 2m”

5ml of 8mol Hydrochloric Acid into the test tube marked “H 8m”

Step 5. Shake the test tubes to get as much flour suspended in the solutions as possible.

Step 6. Quickly add 1ml of 2% amylase solution into each of the test tubes.

Step 7. Add 5 drops of iodine solution into each of the test tubes.

Step 8. Record observations (looking for any colour changes)

Results: During the pre-test, the changes in colour and turbidity give a clear indication of

enzyme activity when added to assorted concentrations of flour.

Pre-test:

Test Tube Amount Of

Flour

Initial Colour Colour After 10

minutes

Turbidity

One 0.5g Light Purple Clear/Transparent Very Low

Two 1g Purple Clear/Transparent Low

Three 1.5g Deep Purple Light Purple Medium

Four 2g Deep Purple Deep Purple Very High

Five 2.5g Deep Purple Deep Purple Very High

Test: Colour changes of solution when amylase activity is exposed to

different levels of pH.

Chemical Permanent Colour

Change

Initial Colour

Change

No Colour Change

8mol Sodium

Hydroxide (NaHO)

2mol Sodium

Hydroxide (NaOH)

Water

(H2O)

Table. 2

Table. 1

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2mol Hydrochloric

Acid

(HCl)

2mol Hydrochloric

Acid

(HCl)

Observations:

Pre-test:

Test tube one with the 0.5 grams initially turned a light/transparent purple

colour and after a couple of minutes, the solution turned clear but still a little

turbid (semi-transparent).

Test tube two with 1g of flour initially turned a soft purple colour and after a

few minutes the solution turned clear but still a little turbid (semi-transparent).

Test tube three with 1.5g of flour initially turned a dark solid purple colour

and after a few minutes it changed to a lighter purple colour which was

moderately turbid. (hazy).

Test tube four had 2 grams of flour which initially turned an extremely deep,

dark purple colour. After a few minutes the solution still was a dark purple

colour which had a very high turbidity (opaque).

Test tube five had 2.5 grams of flour initially turned an extremely deep dark

purple colour. After a few minutes the solution was still a dark purple with

high turbidity (opaque).

In test tubes four and five, there was never a time where all the flour was

suspended in the water.

In all of the test tubes, after the 10 minutes was over there was at least 70% of

the original mass of the flour resting at the bottom of each test tube.

After the 10 minutes in test tube 1, there was a thin layer of purple coloured

flour resting at the bottom of the test tube.

Almost immediate reaction when the iodine solution was added.

Enzyme Test:

The test tube which contained water immediately turned purple.

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The test tubes which contained the Hydrochloric Acid turned a dark colour as

soon as the iodine solution.

As the iodine solution was added to the acids, their turbidity increased

dramatically, there was no light that was able to make it through the solution.

The test tubes which contained the bases had no colour at all throughout the

duration of the experiment.

The bottom 20% of the basic solutions had a yellowish tinge.

After 10 minutes, the acids were still a dark purple/black colour. It had no

significant change since the iodine was added initially.

After 10 minutes, the test tube which contained water had a much lighter

colour than initially.

In both of the test tubes which contained basic chemicals (NaOH), the

turbidity of the mixture increased approaching the bottom of the test tube.

Discussion: Analysis:

The image above shows the success rate amylase had when breaking down the starch

compounds. This pie graph also displays the trends and patterns of results where;

initial change is when the solution went from an initial purple to a more transparent

colour, No colour change is when the solution didn’t change colour at all and

permanent colour change is when the solution turned purple and remained purple

without and colour change. These results were compared to the controlled pre-tests

which were displayed in table.1 which displays the severity of the changes as well.

Green = Base Blue = Water Red = Acid

Diagram. 3

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The results show that test tube which contained the neutral substance (water) enabled

the enzymes (amylase) to work the most efficiently. This is because there was nothing

that caused the denaturing of the enzyme, as a result of this the process of breaking

complex molecules (starch) was able to be carried out as per normal which matched

up with the control tests. 8 tests were conducted in exactly the same manner (to

minimize any introduced variables) which presented identical results for each test.

When starch comes into contract with a strong base such as sodium hydroxide, its

chemical properties change and it turns into a carbohydrate commonly known as

Alkaline-Modified Starch. This means that colour indicators which are supposed to

detect the presence of generic starch will be unable to detect the presence the new

modified starch. Since there was no colour change at all, assumptions can be made

that the sodium hydroxide changed the properties of the starch turning into Alkaline-

Modified Starch. This means it was impossible to track the progress of amylase

breaking down any starch particles. Also, Iodine solution must have stained the

flour while most of it was suspended in the water. This explained the yellow tinge that

was at the bottom of the test tubes which contained the sodium hydroxide.

When starch comes into contract with hydrochloric acid, a carbohydrate called

Dextrin is produced. Dextrin is a form of modified starch but unlike the Alkaline-

Modified Starch, Dextrin is indicated by iodine solution. This explains why the test

tubes containing hydrochloric acid reacted while the test tubes containing strong bases

didn’t. The hydrochloric acid prematurely broke down the starch molecules which

essentially unlocked the Dextrin from the flour grains, this explains why the acids had

an extremely dark colour due to the abundance of Dextrin molecules. But since the

colour never went clear, it is safe to assume that the low pH must have denatured the

amylase enzymes. (Wikipedia : Modified Starch, 2011)

Variables:

The amount of liquid needed to be controlled because any difference in the amount of

space that was available for the reaction takes place could have affected the rate of the

reaction. During the pre-tests there was a constant 10ml of water which was added

into each test tube and during the real testing there was a constant 5mls of chemical

which was added into each test tube. Because the pre-tests were done to provide some

insight into what I should find, the volumes didn’t need to be the same.

Diagram. 4

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The previous diagram shows how the amount of space there is available influences

the rate of the reaction just so long as the amount of amylase and starch in the test

tube remains constant. The more concentrated the solution is, the more contact the

more chance a substrate with attach itself to an active site. Which means the reaction

will occur faster than if the mixture of amylase and starch was less concentrated.

Therefore the amount of flour and amylase that was added into each test tube needed

to be kept constant to have accurate results. (Yahoo Answers: How does the amount of

enzyme effect enzyme reaction rate?)

Also the temperature was kept constant throughout all of the testing samples to ensure

that heat was not a complicating factor in any of the reactions which need to be taken

into account when compiling data.

The independent variable was the difference of pH in the different test tubes, it was

the only thing that was changed throughout the duration of the experiment. The reason

for this was to ensure than there were no other factors involved in the rate at which

the amylase breaks down the starch.

The dependent variable was the colour change in this experiment, the controls showed

what a successful test looks like. The colour change was the sign that enzymes were

doing their job correctly. Since the rate at which the enzymes work is shown by the

colour change, and the rate the enzymes work is influenced by the pH in its

environment. The colour change is dependent on the pH (the independent variable) in

the environment which surrounds the enzyme (eg. Sodium Hydroxide, Water and

Hydrochloric Acid).

Suggestions for improvement:

The main problem with the experiment which was never addressed was that the bases

and acids were too strong (Sodium Hydroxide has a pH of 14 and Hydrochloric Acid

has a pH of 0). This would have denatured the amylase almost instantaneously upon

contact, which left them no time for the reaction to take place. It would have been

better to use weaker bases and weaker acids or even to test in more pH environments

to get a wider spread of data. Previous studies have proven that amylase from human

saliva is most efficiently between a pH of 5 and 9 (the table below displays the results

from the research). (Reactions and Enzymes, 2010)

The table adjacent represents enzyme activity vs pH. Taken from : http://www.emc.maricopa.edu/faculty/farabee/BIOBK/biobookenzym.html (accessed 15/9/2011)

Diagram. 5

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Another problem which was encountered during the experiment was the fact that flour

is not a water soluble substance. This means that the flour grains stay suspended in the

water for a short amount of time. A suggestion to improve this would be to use a

source of starch where each grain is much finer, such as rice water. Also, to maximize

the amount of flour suspended, the method for getting the flour suspended could also

be changed.

The image above shows that turning the test tube on an angle will increase the surface

area of the flour that comes into contact with the chemical that is poured. Therefore

there will be more starch suspended in the liquid.

Conclusion: The purpose of this investigation was to determine the effects of different pH on

amylase enzymes. Compared to the control, there was evidence that the pH of the

solution in the test tube did have a major influence in the rate in which the reaction

occurred. It was proven that extremes in the pH slow the rate the amylase breaks

down starch. This was due to the potency of the chemicals being used which resulted

in whole lot of complicating factors which were introduced into the experimental side

of the investigation which means the original hypothesis was neither accepted nor

denied. Although each test tube showed difference compared to each other and the

results were consistent though out the experiment, there was not enough evidence

given by the experiment to propose a valid conclusion to the experiment. Further

testing with a wider range of data is required to completely determine the differences

in the rates of reaction in different pH in an environment.

The data gathered from this experiment could have real life implications for patients

who have enzyme deficiencies. For example, a patient who is experiencing low

energy levels might have an amylase deficiency from not having enough

carbohydrates being broken down in the body. Amylase deficiency also related to

other complications such as inflammation, allergic reactions, eczema, psoriasis and

atopic dermatitis. (Buzzle: Amylase Levels in the Blood, updated 2011)

Diagram. 6

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Bibliography:

Wikipedia, Unknown Author. 2011. http://en.wikipedia.org/wiki/Enzyme [accessed

30/7/2011]

This Wikipedia article introduces the basics about enzymes and their role in the

human body.

The aim for the article was to provide the reader with a basic knowledge of biological

catalysts and the author uses results found in other studies and images to entice and

help the reader understand.

The scope of article was only to provide a basis of what is and how enzymes work.

This article was extremely useful to my research because it provided enough

information about enzymes for me to perform the experiment and form a suitable

hypothesis.

The only limitation to this article was the fact that most of the information that was

given was irrelevant to my investigation. This article provided an adequate amount of

information to complete the understanding of how enzymes work, more research is

required to show how they react when exposed to different conditions.

Wikipedia:Enzymes formed the basis of the investigation.

Beta Force: What are Enzymes and Why We Need Them, Unknown Author. 2006.

http://www.beta-glucan-info.com/enzyme_facts.htm [accessed 30/07/2011]

This internet article focuses purely on what enzymes do in our body.

The author uses simple theories and explanations to all for more accessibility to the

article.

The purpose of this article was to inform the on basics on how enzymes work in the

human body.

This article was useful to the investigation because it gave enough information about

the activity of enzymes to tie in with the aim of then experiment.

The only limitation to this article was that it might have been too brief and the

information can be found in greater depth and technicality on other sites. Although

this article provides only a small portion of the understanding, it was a necessary part

of the research.

Brain, Marshall. "How Cells Work" 01 April 2000. HowStuffWorks.com.

<http://science.howstuffworks.com/environmental/life/cellular-microscopic/cell.htm>

[accessed 1/08/2011.]

This article is basically a small glossary which contained anything relevant to

enzymes.

The author doesn’t introduce any complex ideas throughout the whole article, only

defines certain terminology.

The purpose of this article was to get a quick idea of what something means (such as

“inhibitors”)

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The major limitation to this article what that there was very little actual information

on the processes of enzyme reactions. Although it did not make up the bulk of the

research, it provided a good quality reference point for definitions.

Spenceley M, Weller, B. Mason, M, Fullerton, K. Tsilemanis, C. Evans, B. Ladiges,

P. McKenzie, J. Batterham,P. Pearson Education Australia, 2004. “Rate of Enzyme

Reaction” Heinemann Queensland Science Project Biology: A Contextual Approach,

Melbourne., pp 168-169.

This text book was created to give senior school students a vivid understanding of all

the aspects in biology.

The authors kept in mind the intended audience for this text book, so they used both

complex and simple explanations for theories as well as images and diagrams to

increase the ease of learning a new concept or idea.

This book was extremely useful as it provided information about all the aspects of this

investigation and the sources are trustworthy.

The only limitation to this book was that there was nothing that explained anything

about the chemistry side of enzymes. Although nothing was taken out and used in the

introduction, it did serve as a good reference point for everything in the introduction.

Other References:

Unknown Author. 2007, http://lugolsiodinesolution.com/ [accessed 25/8/2011]

Unknown Author. 2008,

http://answers.yahoo.com/question/index?qid=20080914115624AADqZSQ [accessed

25/8/2011]

Sandhyarani.N. 2011, http://www.buzzle.com/articles/amylase-levels-in-blood.html

[accessed 26/8/2011]

Unknown Author. 2010,

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/biobookenzym.html [accessed

26/8/2011]