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Enzymes Experiment

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Enzymes Experiment


Introduction

Enzymes are biological catalysts that promote some of the thousands of
chemical reactions that occur in living cells. They are large
molecules that have a unique three-dimensional structure that allows
it to react with a specific substrate. It is hypothesized that pH
levels out of the normal range found in cells would denature the
enzyme, slowing the enzyme's reaction rates.

Hydrogen Peroxide (H2O2) is a poisonous chemical that is continually
being formed as a byproduct of reactions in peroxisomes of living
cells. Since it is poisonous, the cells must either rid themselves of
it or change it to something that is not harmful to the body, the
consequence of not doing so, ultimately being death. The enzyme
catalase is found in animal and plant tissues, and is especially
abundant in the liver. Catalase reacts with the H2O2 to form water and
oxygen:

2 H2O2 + Catalase à 2 H2O + O2 + Catalase

The amino acids forming the enzyme contain functional groups such as
carboxyl groups and amino groups. These functional groups are able to
react with excess H+ ions in solution, resulting in the disruption of
the enzymes' structure. Most enzymes, for example amylase, have an
optimum pH in the neutral pH range that resembles that of the cell
environment, usually being the neutral pH level of 7. However there
are exceptions depending on the environment that the enzyme must work
in. For example, pepsins, which are protein digesting enzymes found in
the stomach, have an unusually low optimal pH of 2.5.

In this experiment, catalase will be extracted from sheep liver. The
extracted enzyme will be mixed with a solution of known pH. The
reaction time of the enzyme will then be measured and graphed.

It is expected that the catalase would perform the most effectively,
with the quickest reaction time, in a pH range of 7, as that is the
physiological pH of most cells in the human body. It is also expected
that the reaction time will rise as the pH is raised or lowered away
from 7.

Apparatus

- Sodium Hydroxide (NaOH (aq)) of 0.2M

- Hydrochloric acid (HCl (aq)) of 0.1M

- Concentrated Hydrogen Peroxide (H2O2 (aq))

- Universal Indicator

- Tap Water

- Sheep Liver

- Sand

- Mortar and Pestle

- Evaporation Dish

- 5 Measuring Cylinders

- Large glass pipette and pipette filler

- Small glass pipettes

- Plastic pipettes

- Glass stirring rod

- Tweezers

- Filter Paper

- Stop Watch



Method
======

(1) A small portion of sheep liver was ground up using a mortar and
pestle and sand to act as abrasive matter. A small amount of tap water
was added so that a dark red liquid was formed. This liquid was then
poured into an evaporation dish and set aside.

(2) 80mL of tap water was poured in each measuring cylinder. 4 drops
of Universal Indicator was added to each cylinder.

(3) To two cylinders, drops of HCl were successively added until pHs
of 2 and of 4 were obtained. The solutions were coloured red and
orange respectively. To another two cylinders, drops of NaOH were
added until pHs of 9 and of 11 were obtained. The solutions were
coloured turquoise and navy blue respectively. No acid or base was
added to the final cylinder: it had a pH of 7 and was coloured lime
green.

(4) Using a pipette, solution was removed from cylinders that
contained more than 80mL (due to the adding of base and acid) to bring
the volume of each solution back down to 80mL.

(5) 1mL of H2O2 was added to each cylinder and stirred.

[IMAGE]

(6) Using tweezers, a square of filter paper (1cm2) was saturated with
the dark red liver solution. It was subsequently immersed into the pH
solution of 11 and left to float to the bottom. The stopwatch was
started as soon as the paper was immersed and stopped as soon as it
floated back up to the top.

(7) The time taken was recorded and this process was repeated for all
cylinders.

(8) The entire experiment was repeated again.



Key Variable
============

All experiments were conducted at room temperature, the only variable
being the pH of the solution that the Hydrogen Peroxide and the filter
paper were put into.


Results
-------

The first experiment was unsuccessful and admitted no results as it
was found that the Hydrogen Peroxide used was faulty. Therefore the
first trial will not be recorded or graphed. The following table
records the results of the subsequent experiment in which the Hydrogen
Peroxide was effective.

Table 1: Reaction times collected from the experiment for varying pH
levels.

pH Level of Solution (in pH)

2

4

7

9

11

Reaction Time (min:ss)

4:48

1:17

1:28

2:33

3:04

Graph 1: Data from Table 1 [IMAGE]

As seen on the graph, reaction times were the fastest at an acid pH of
4. Reaction times rose dramatically for catalase in a strong acid pH
of 2. Reaction times also rose for reactions carried out in basic pH
solutions.

The pH of liver was recorded as 5.5.


Discussion
----------

The purpose of this experiment was to investigate the effects of pH on
catalase activity. Since the enzyme was thought to usually be found in
a fairly neutral environment, it was hypothesized that the enzyme
activity would be slowed or stopped in pH solutions that were either
acidic or basic. It was expected that the reaction rate of the enzyme
would be the highest in a solution of pH 7. It was also expected that
the reaction rate would decrease as the pH deviated from 7. This was
due to the observation that the physiological environment in which
most cells are placed have a neutral pH.

The data indicated that the catalase worked most effectively in pH of
4, however it took only 11 seconds longer in the neutral solution.
This is only a small margin that could be explained by many reasons.
There is a possibility that the experiment carried out for a pH of 7
was compromised by slightly different conditions. When the filter
paper was dipped into the liquid liver solution, a fragment of liver
could have attached itself to the filter paper meaning that its weight
was greater and thus it could been more difficult to rise to the top
again. There could have been a slight miscalculation of the amount of
H2O2 put in the pH 7 solution.

Another possible explanation is that catalase works more efficiently
in slightly more acidic solutions. As liver was found to have a pH of
5.5, this being slightly acidic, this is a valid conclusion.

The data indicated that catalase works very inefficiently in a pH of 2
(strongly acidic). The amount of H+ in solution interfered greatly
with the structure of the enzyme causing denaturation.

Although the basic solutions permitted the catalase to carry out its
task eventually, it was not as efficient as the neutral or weak acidic
solutions taking approximately double the time to react.


Errors and Improvements
-----------------------

There are some ways in which this experiment could be improved. As the
first trial proved, it is essential to ensure that the chemicals used
are not stale. As measurements for the different amounts of chemicals
used, were judged by the human eye, there could have been
inaccuracies. Sometimes the same stirring rod was used for both the
acid, basic, and neutral cylinders, perhaps resulting in a 'neutral'
control solution being slightly acidic or basic.

To further this investigation, there are many potentially interesting
experiments. Essentially, repeating the temperature but with more
variation of pHs between 5 and 8 would lead to ascertaining the exact
optimal pH for catalase in the liver. Also, one could test the impact
of changes in temperature and enzyme concentration on reaction time as
well as testing other enzymes on their substrate to examine whether
comparable results were found. The most important thing is to repeat
the experiment to ensure that there were no problems such as
impurities or miscalculations.


Conclusion
----------

The hypothesis was rejected. Enzymes work most effectively in slightly
acidic solutions (ie, pH 4) and their efficiency decreases as the pH
of a solution neutralises or becomes more acidic or more basic. This
is due to the fact that the liver is slightly acidic, and enzymes work
best in the environment to which they are accustomed.

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