Core Practical 4: The effect of enzyme concentration on rate of

reaction.

Aim: To investigate how enzyme concentration can affect the initial rate of reaction.
Background Information:
Enzymes are proteins which act as biological catalysts. They speed up the rate of reaction by
lowering the activation energy. Enzymes are highly specific which mean only one substrate can
bind with a specific enzyme. It has an active site which is the part of the enzyme that the
substrate binds on to. Enzymes work at a certain pH and at a certain temperature. The enzyme
would denature once it has reached its optimum pH or its optimum temperature. The substrate
concentration also affects the rate of reaction. In the case of this experiment the protein
substrate that is being used is casein this is found in milk and is the protein that makes the milk
cloudy. The enzyme trypsin breaks down the protein to a polypeptide. Trypsin is an enzyme
which is found in the small intestine and is produced in the pancreas. These enzymes work best
in alkaline conditions. By increasing the enzyme concentration, the rate of reaction also
increases. In this experiment, we measure the initial rate of reaction because as the reaction
goes on, the substrate concentration decreases and the amount of product would increase.
Therefore, the chance of a substrate bumping into an enzyme also decreases so eventually the
rate of reaction would also decrease.

Hypothesis:
The higher the enzyme concentration, the faster the enzyme: trypsin will be broken down, so
the faster the rate of reaction.

Independent Variable:
Protease concentration (measured in percentage)
Dependent Variable:
Time taken for enzyme to breakdown substrate (in seconds)
Control Variables:

 Temperature
 Volume of enzyme solution
 Volume of substrate
 Concentration of substrate

Equipment:
 Range of trypsin concentrations (0%, 1%, 2%, 3% and 4%)
 Casein/Milk Powder solution
 Stopwatch
 Test tubes
 Test tube rack
 Distilled water
 Pipettes

Method:

1. Pipette 2cmᶟ of distilled water into a cuvette

2. Place this cuvette into the colorimeter in order to calibrate the colorimeter.
3. Pipette 2 cmᶟ of milk powder solution into another cuvette.
4. Pipette 2 cmᶟ of the trypsin solution into the cuvette starting with 0%. Mix thoroughly
and immediately put this cuvette into the colorimeter and start the stop clock.
5. Measure the absorbance at suitable time intervals for 1.30 minutes or until there is little
change in reaction.
6. Repeat these steps with the different trypsin concentrations (1%,2%,3%,4%) making
sure to calibrate the colorimeter with a cuvette of water after each of the
concentrations.

Risk Assessment:

During this experiment there were no serious risks. There were some minor risks such as the
fact that the enzyme solution could spill on the floor and this could be a risk to anyone around
the area as someone may slip and fall. In order to prevent this, you can make sure that all the
solution is placed in the centre of the table ensuring that it doesn’t drop and spill also it should
cautiously when moving around with it so you don’t drop the solution. Another risk may be if
any glassware used drops and shatters. This can cause harm to anyone near the glass, they may
get cut and hurt themselves. In order to prevent this, you should make sure that the glassware
is kept in the middle of the table so there is a lower risk of it dropping and smashing.

Average Absorbance
Trypsin 0 sec 15 sec 30 sec 45 sec 60 sec 75 sec 90 sec
concentration

0% 1.90 1.90 1.90 1.90 1.90 1.90 1.90

1% 2.01 1.57 1.21 0.84 0.47 0.30 0.11
2% 2.01 1.42 0.87 0.41 0.14 0.10 0.06
3% 2.01 1.39 0.75 0.23 0.13 0.11 0.09
4% 2.01 1.38 0.68 0.19 0.19 0.16 0.13
Analysis of results:

When taking down the results it became evident that as the enzyme concentration increased, which in
this case is the trypsin concentration the absorbance had also decreased. This shows that as the
concentration of trypsin increased the milk powder solution had become less cloudy and more visible
because the absorbance slowly decreased through the different concentrations.

At the trypsin concentration of 4% we can see that the average absorbance was the smallest as it was
1.38 at 15 seconds this shows that the rate of reaction is faster with a higher concentration of enzyme
concentration this is because there are more enzymes so therefore more active sites in order to
catalyse the substrate therefore there are more successful collisions in a higher concentration
so the rate of reaction increases. At 45 seconds and 60 seconds of the absorbance of 4% of
enzyme concentration the absorbance stays the same at 0.19, because it stays the same for two
different time intervals this could show to us that it may have reached its optimum.

The 4% trypsin differs to the 2% enzyme concentration as the average absorbance is much
higher at 2% for example at 30 seconds it is 0.87 whereas at 4% for 30 seconds it is 0.68. As the
absorbance for the enzyme concentration is much higher in 2% it shows to us that as the
concentration of the trypsin is lower there are therefore less enzymes so less active sites for the
substrates to bind on to. This means that at 2% the milk solution would be cloudier because the
enzyme hasn’t been able to fully break the solution down.

Evaluation:

One limitation of this experiment is that we could’ve carried out more repeats of the
experiment this would’ve allowed us to obtain more valid and reliable results. By repeating the
experiment, it is easier for anomalous results to stick out and then is easier to discount the
anomalous results. This increases validity and accuracy of the experiment.

Another limitation of the experiment is that it can be difficult to calculate the absorbance
because the colorimeter may not always work. This was a problem for me during this practical
because throughout the practical the colorimeter wouldn’t provide results but we still managed
to gain results as it still worked throughout most of the solutions. This is a limitation because it
can reduce the accuracy of the results.

Another weakness of the experiment is that if you don’t calibrate the colorimeter after each
different concentrations this can reduce the accuracy of the results. This can also cause the
colorimeter to give inaccurate results for the other concentrations. To prevent this, you should
make sure that you calibrate the colorimeter before each other trypsin concentrations.

One limitation of this experiment is that you need to make sure that when you pipette the milk
solution into the enzyme solution you need to make sure that you put it in the colorimeter
immediately because it may lead to inaccurate results as the reaction would’ve already started
once the two solutions make contact.
Conclusion:

From the results, it is possible to conclude that the hypothesis that we had created was proved
right as it was that, as the concentration of the trypsin solution had increased, the rate of
reaction also increased. A higher concentration means more of the solution is made up of
enzymes, so the reaction will happen quicker and at a faster rate if more enzymes are present.
Overall, the higher the concentration, the more enzymes that are present in the mixture so
more reactions can take place as the enzymes will have more reactions, leading to a higher
amount of successful collisions, which would increase the rate of reaction. Therefore, overall the
experiment was successful as we proved the hypothesis and the results reflect the hypothesis
we had begun to prove.

Bibliography:

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sjhoward.co.uk » Effect of Enzyme Concentration on rate of Hydrolysis of gelatin by protease enzymes. 2018.
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