Investigating the effect of enzyme concentration on the rate of an enzyme catalysed reaction

Investigating the effect of enzyme concentration on the rate of an enzyme catalysed reactionAims: This is an experiment to investigate how the concentration of
the substrate Hydrogen Peroxide (H2O2) affects the rate of reaction of
the enzyme Catalase, in yeast cells.Catalase product

The formula: 2H2O2 2H2O + O2

Background information: Enzymes such as catalase are globular protein
molecules found in all living cells. They are biological catalysts,
and are used to speed up a specific reaction rate within the cell.
They reduce the level of activation energy needed in a reaction, which
speeds up the rate of a reaction. The higher the activation energy,
the slower the reaction will be. The two graphs below show the amounts
of activation energy needed with and without an enzyme:

All enzymes contain an active site, a depression in the enzyme
molecule, where a specific substrate molecule can fit into exactly and
bind to. All enzymes have a substrate molecule, which is the same
shape as its active site. When the substrate binds to the enzyme?s
active site, it forms an enzyme-substrate complex, which produces the
product(s). Enzymes only perform one type of reaction, and only have
one specific type of substrate to do this.

Catalase is an enzyme found in food such as potato and liver, and is
used to remove Hydrogen Peroxide from cells, as it is the poisonous
by-product of metabolism. There are two types of enzyme-substrate
metabolic reactions: anabolic and catabolic. An anabolic reaction is
when there is a two-part substrate which is built together to form a
new molecule. A catabolic reaction is where one substrate is broken
down to produce two products. The two diagrams show both of these
reactions which show the lock and key theory:

An anabolic reaction:

A catabolic reaction:

There are four factors that can affect the rate of an enzyme reaction.
They are:

o The pH
o The temperature
o The concentration of the enzyme solution
o The concentration of the substrate solution.

The pH can affect the rate of an enzyme-catalysed reaction by
affecting the bonds in the secondary and tertiary structure of the
enzyme?s active site. If the bonds are broken, the shape of the active
site can be distorted, so the substrate molecule cannot bind to the
enzyme causing no enzyme-substrate complexes, and therefore producing
no products. Each enzyme has an optimum pH. Most enzymes work best in
neutral or slightly alkaline conditions.

The temperature affects the rate of reaction. If you increase the
temperature you increase the amount of energy, therefore more enzyme
and substrate molecules will collide more often, increasing random
movement. The more collisions there are the higher the chance is that
a substrate molecule will fit an active fit. Each enzyme has an
optimum temperature; this is where the rate of reaction is at its
maximum. If the temperature is above the optimum, the rate of reaction
starts to decrease as the active site can start to distort, causing no
enzyme-substrate complexes and producing no product, as the substrate
cannot bind. At very high temperatures the number of collisions may be
very high but without the correct shape of the active sites, no
products can be formed. At these very high temperatures the enzyme can
become denatured. This means that the enzyme?s active site has become
permanently damaged.

The concentration of the enzyme solution also affects the rate of an
enzyme reaction. If there are more enzyme molecules than substrate
molecules then the substrate would be a limiting factor, as there
would not be enough substrate to continue forming products. When all
the active sites are in use, the optimum rate will have been achieved.
But the reaction will take place very quickly and then finish if no
more substrate is added because the substrate will run out.

In the same way the concentration of the substrate affects the rate of
reaction, only in this case the limiting factor is the lack of active
sites. The rate of this reaction will be slow as there are not enough
active sites. The optimum rate occurs when all of the active sites are
in use.

We will be investigating the effect of enzyme concentration.

Prediction: I predict that the greater the concentration of catalase,
the faster the rate of reaction. If yeast is the enzyme then the
greater the concentration of yeast, the greater the volume of oxygen
(the product) is in the initial rate (the first thirty seconds). This
is because if the concentration of the catalase is high. There are a
lot of enzymes in the solution, and therefore a large number of active
sites available. This means that there is a greater chance of a
substrate colliding with an active site, as there are more active
sites available. I will only be measuring the rate of reaction during
the initial rate as after a period of time the substrate will run out,
and the rate will decrease. The product produced from the
enzyme-substrate complexes is oxygen, Therefore, the greater the
volume of oxygen is in the initial rate, the faster the rate of the
reaction.

This prediction could be quantitative. If I double the yeast
concentration I should get double the volume in the first thirty
seconds. (The initial rate.)

Variables:

o My independent variable will be the enzyme (yeast) concentration. I
will vary this using serial dilution, in order to make up the yeast
concentrations. The five yeast concentrations I will use are 0.1%,
0.25%, 0.5%, 0.75%, and 1.0%. In order to make these concentrations I
will make up 1.0% by having 1% catalase and 99% distilled water. Then
I will half the concentration of to make 0.5%. I will half this to
make 0.25%. In order to make 0.75% I will use ? of the concentration
of 1.0%. But I will keep the volume constant.

o My dependant variable will be the amount of oxygen released.

The variables that I will need to control will be:

o The pH
o The temperature
o The substrate concentration
o The volume of substrate added
o The volume of yeast added

All of these control variables will need to be controlled by me, and
to be kept the same throughout my experiment, in order to keep it fair
and for the results to be accurate. I am only testing the effect of
enzyme concentration on the rate of reaction so the other factors will
have to remain the same, so that they do not affect my results.

To get accurate measurements of the enzyme and substrate I will use an
automatic pipette. I will keep the volume of the reaction mix constant
so it will not affect the rate. To control the temperature during the
experiment I will keep the enzyme and substrate solution in a
waterbath set at 30?C. To control the substrate concentration I will
use the same volume and concentration each time, so that it will not
affect my results. I have decided to control the pH, and keep it the
same so that this will not affect the rate of reaction using a buffer.

Preliminary study: To try and get an idea of what sort of method and
apparatus I should use I consulted a number of biology texts. To
measure the rate of reaction by the amount of oxygen given off, there
were several different methods. One method (of which there were
several variations) shows how the oxygen could be measured by
delivering the gas through a rubber delivery tube. They measured the
volume of oxygen produced by counting the number of bubbles produced.
The higher the number of bubbles produced in thirty seconds meant a
faster reaction rate. A diagram of this method is shown below:

I have decided not to use this method, as it is a very inaccurate way
of measuring the volume of oxygen given off, as the size and volume of
bubbles vary. However, using a syringe to add the yeast solution is a
very good idea as it is easy to use and no oxygen will be lost.

I gained a more reliable method where the oxygen was measured using a
measuring cylinder. This is a good idea but only gives the volume of
oxygen produced up to every 1cm3. A burette would be more accurate as
it measures every 0.1cm3, however this would be difficult to use, and
there could be a risk of loosing some oxygen as I put the burette
below water and seal. But using the thistle funnel through the bung is
a good idea, as no gas can be lost because it is not necessary to
remove the bung to add the yeast to the hydrogen peroxide.

From another source I have got the idea of collecting the oxygen in a
gas cylinder instead of a measuring cylinder or burette. This would be
easy to use and should not loose much of the gas, as it is tightly
sealed and smooth as it is made from glass.

Pilot study: I carried out a pilot test in order to find out what
volumes, quantities and concentrations of hydrogen peroxide to use. To
do this I set up the apparatus as shown in the diagram below:

I set up these apparatus so that I could decide what concentration of
H2O2 and what quantities to use, and to see what would give me decent
results in the first thirty seconds. Firstly I used 5cm3 of yeast with
0.1% concentration, and 10cm3 of H2O2 with a concentration of 1 molar.
(To make the concentration of yeast you dilute with distilled water.)
But this did not give out very good results, as it produced little or
no oxygen. Therefore I increased the quantities of H2O2 and yeast, and
increased the concentration of H2O2. I finally got decent results when
I used 40cm3 of yeast with a concentration of 0.1% with 10cm3 of H2O2
with a two molar concentration. I couldn?t increase the concentration
of hydrogen peroxide, as two molar H2O2 is the strongest concentration
of hydrogen peroxide legally allowed in schools. I also didn?t want to
use too much of hydrogen peroxide but not too little either. With a
higher volume of yeast the results were improving, as more oxygen was
being produced. For my pilot study I only used yeast with a
concentration of 0.1%, because if oxygen was produced with a low
concentration of yeast, then I knew that it would with a higher
concentration of 1.0%. From my pilot test I have decided to use
40cm3of yeast and 10cm3of hydrogen peroxide with a two molar
concentration as using these quantities and concentrations, oxygen has
been produced during the initial rate. During the pilot study I
realised that the syringe would not hold 40cm3 of yeast, therefore

graph
However, I have realised that this method involves removing the bung
to add the hydrogen peroxide and the yeast. This could result in
loosing some oxygen whilst we put the bung back in the conical flask.

Therefore, from the range of methods I have seen I have decided to use
a side-arm conical flask connected to a gas syringe. To add the yeast,
my original idea was to use a syringe, although it would not hold 40ml
of yeast. Therefore I will put the yeast in the conical flask with a
buffer and then add the hydrogen peroxide using a syringe through the
bung. I have also realized that I have not controlled the temperature.
Therefore I will keep the conical flask in a water bath set at 30?C
throughout the experiment. A final diagram of the apparatus I am going
to use is shown below:

Apparatus:

Ø Stand
Ø Clamp
Ø Boss
Ø Bung cut into two
Ø Conical flask with side arm
Ø 10cm3Hydrogen peroxide (two molar)
Ø 40cm3 of Yeast
Ø Buffer
Ø Automatic pipette
Ø Gas syringe
Ø Beakers
Ø Goggles for eye safety
Ø Laboratory coat to protect skin and clothes
Ø Stop clock
Ø Syringe
Ø Water bath set at 30?C.

I have chosen the stand, clamp and boss to hold the gas cylinder,
horizontally in the air to be sure that it is not affected by any
variables. The conical flask and the bung is used to hold the enzyme
solution and the substrate, also to stop any gas from escaping, as we
are measuring the oxygen gas. The bung will be cut in half to allow
the syringe holding the hydrogen peroxide to penetrate. The conical
flask has a side arm that allows the gas to be transported to the gas
syringe via the delivery tube. The rubber delivery tube is used to
transport the gas to the gas syringe to be measured and to minimise
the amount of oxygen lost. The buffer will control the pH of the
yeast. The water bath will control the temperature. I will use an
automatic pipette, as it is very accurate and precise as it measures
every m1000th mil. I will use a gas syringe to measure the oxygen
produced. It is specially designed to measure gas and is made from
smooth glass, which will allow all the gas to be collected and easily
measured. This makes it very precise and accurate and is easy to use.
The stop clock is used to time the initial rate. However an
individual?s reaction rate varies, so is not very accurate, but is the
easiest way. I will be required to wear a laboratory coat and goggles
to protect my eyes, skin and clothes as hydrogen peroxide, and some
enzymes can be hazardous and harmful.

Risk assessment:

Hydrogen peroxide can be corrosive if strong, and an irritant to the
skin and eyes. It should not be swallowed. It can corrode clothes and
irritate the skin if not washed off thoroughly. Eye protection should
be worn and should be handled with care. Contact with the skin should
be avoided and clothing should be protected. You should also mop/clean
up any spills. All enzymes are potential allergies and can irritate
membranes in the eyes or nose (if inhaled). They may also cause an
asthmatic attack. Eye protection should be worn and care should be
taken when handling. You should avoid handling, inhaling or contact
with the skin wherever possible. They must not be swallowed and spills
must be mopped up immediately.

Method:

Set up apparatus and water bath. Care should be taken to avoid any
?gaps? where the oxygen could escape.
ii. Measure out 10cm3* of hydrogen peroxide (two molar) using an
automatic pipette for accuracy. Carefully* pipette into the syringe.

iii. Measure out 40cm3* of yeast at a concentration of 0.1% using a
clean automatic pipette. Carefully* add the yeast solution to the
conical flask.

iv. Carefully* add the hydrogen peroxide solution.

v. Time for 30 seconds* using a stop clock, and record in a table the
volume of oxygen produced.

vi. Repeat 3 times using increasing concentrations of the yeast
solution. (0.1,0.25,0.5,0.75,1.0)

(*to be carried out very accurately.)

graph
The filter paper rises because it is placed in the yeast solution, and
then dropped into the hydrogen peroxide solution. My prediction would
suggest that the higher the yeast concentration is, the faster the
filter paper should rise. This is because there would be more enzymes;
therefore there would be more active sites available to make
enzyme-substrate complexes with, producing oxygen faster. The faster
oxygen is produced, the more bubbles are produced and in theory, the
filter paper should start to rise, as the bubbles lift it up to the
surface.

Conclusion: My results tell me that the higher the concentration of
yeast, the faster the reaction, because the faster the filter paper
rose. The table shows me that with a 0.02% concentration of yeast, the
filter paper rose to the top of the flat-bottomed tube of hydrogen
peroxide in a mean time of 71.22 seconds. With a higher concentration
of yeast, at 0.04%, the mean time taken for the filter paper to rise
was lower, at 36.48 seconds. These two results show a large difference
in the time taken for the filter paper to rise. In fact, the result is
just over half what the previous result was. This is evidence that
this study is quantitative, because as If I double the concentration,
I get on average, almost double the time.

With the yeast concentration at 0.06%, the time taken had lowered down
to a mean time of 22.13 seconds. It had decreased further with a
higher enzyme concentration of 0.08%. At this concentration there was
a mean time of 15.83 seconds. This shows that the rate of reaction was
speeding up. Finally at the highest concentration of 0.1% the filter
paper had taken a mean time of 10.69 seconds. This proves that the
rate of reaction was faster with a higher concentration, because at
low concentrations the filter paper took a longer time to rise than it
did with higher concentrations. This can be seen in my table and my
graph. The graph shows a curve. At first the drop is rapid between
0.02% and 0.04%, as the gradient is steep, but then the drop becomes
less steep, as the drop becomes more gradual. You can start to see
that the graph is starting to level off as the concentration
increases. It would seem that if I were to extend the curve, it would
level off completely at a concentration of around 0.15%. The reaction
rate would then become constant due to other factors.

Interpretation: My conclusion shows that the greater the concentration
of catalase, the faster the reaction rate. I can explain this because
if there is a higher concentration of yeast there will be more active
sites present and available to bind with a substrate to form an
enzyme-substrate complex. The reaction rate will speed up because if
the number of enzymes increases, there is more chance that a substrate
will collide with the enzyme and fit into its active site. Therefore
there would be an increased chance of a hydrogen peroxide molecule
binding with a catalase molecule, producing an oxygen product. The
increased rate of reaction will mean that more oxygen is formed. This
increase in productivity of oxygen will form more oxygen filled
bubbles. These bubbles will lift the filter paper to the surface. So
basically, the faster the rate of the reaction means more oxygen,
which means more bubbles, which makes the filter paper rise faster.
Therefore, the faster the filter paper is lifted, the faster the rate
of reaction. This proves that my prediction is correct, as the results
match my prediction, which is backed up by scientific knowledge.

Evaluation:

The experiment went well as most of the results matched my prediction.
However, I have noticed that there appears to be one anomalous result.
This can been seen in the table by looking at the third repeat for the
0.02% concentration of yeast. This result is very different from the
other two attempts, as the result is 81.00, whilst the other results
are around the mid-sixties. I can see that there is a big difference
between the results as there is a large standard deviation. The
standard deviation tells me how much the results deviate from each
other. As you can see in the table the higher concentrations tend to
have a low standard deviation. This suggests that all three attempts
are similar. However, the two lowest concentrations have higher
standard deviations. This is because the results are more spread out,
or there is an anomalous result. The standard deviation is required to
see how reliable the mean result is. If there is a high standard
deviation, then the mean result will not be as accurate, as one of the
three results deviate from the norm. This anomalous result will affect
the mean result. In this case the means have increased due to the
anomalous result. This makes the mean time different from the other
two results, despite being the average the results may not be as
accurate as it could be. To overcome this problem you could do more
repeats, and then find the mean. The 0.04% of yeast concentration has
a slight anomalous result in the third attempt, but is not so large as
the result for the 0.02% concentration. Two things may have caused
these two anomalous results, either the accuracy, or the technique.

For accuracy I tried to be as accurate as possible, however there were
some things I didn?t control. The results might not have been as
accurate as they could have been when timing. I only used a stop
clock, which relied on my reaction, to stop the clock. Everyone?s
reaction times are different so is not particularly the most accurate
way of timing. Also you had to decide where you would start timing.
Would you start timing as soon as it touches the bottom or when it is
dropped onto the surface? This arises another problem with my method.
I didn?t measure the height I dropped the filter paper from. This
could make the results slightly inaccurate, as if I was timing from
when it hit the surface, the speed that it drops may differ slightly.
Another problem with the timing was that every attempt was different.
Sometimes the paper did not rise in a flat position. Sometimes the
paper would start to rise then turn on its side and fall again, in
which case you would have to keep on timing, as the result may be
inaccurate. If this happened then when would I stop the clock? Should
I stop timing when it is flat on the surface or if it is on its side
at the top?

The other problems were with the technique. The size of each filter
paper square were not exactly the same. Some were slightly different
sizes, and some were slightly thicker than others. This could be a
problem because if the paper was too big then more of the enzyme could
be absorbed. Therefore more oxygen could be produced and there could
be a faster rate of reaction. This would also be inaccurate. Another
problem was that I didn?t time how long I left the filter paper in the
yeast solution. This could also have meant that more of the enzyme
could be absorbs, so more oxygen may be produced causing a faster
reaction rate. To overcome this problem I could time how long the
paper was left in the yeast solution. However, this would cause
further problems with accuracy, as everyone?s reaction times are
different.

Overall I feel that the biggest problem was the accuracy when timing.
The paper would sometimes turn onto its side and fall, before it
reached the surface. Also it was hard to decide whether to use the
results when this happened. To improve this problem you would need to
use something more accurate than a stop clock. Something that stops
timing once it reaches a certain point. You would also need to stop
the filter paper from turning onto its side and falling. You could
something to keep the paper stable. If we used a different shape then
it could not turn onto its side. Maybe a sphere or a cube instead of a
square of filter paper.

If I were to do this experiment again I would take into account all
the problems I have noticed here. I will also try to be more accurate.
However despite these criticisms I do feel that my conclusion is
reliable, as I only had two anomalous results, and it does support my
prediction.

Investigating the effect of enzyme concentration on the rate of an enzyme catalysed reaction 8.5 of 10 on the basis of 3720 Review.