The effect of enzyme concentration on enzyme activity.
Aim
To investigate the effect of an increase in concentration of protease* on the rate of hydrolysis of
the milk protein, casein.
* The protease that is used in this investigation is trypsin
Rationale and background:
The pancreatic duct in cystic fibrosis sufferers frequently becomes blocked by excess sticky
mucus, which reduces and can prevent the release of pancreatic enzymes into the small intestine.
Continue … and explain the reason why this experiment is set!
Milk powder contains a white protein called casein. When protease is added to white suspended
milk powder it turns clear because …………….(good explanation that you can repeat - hopefully –
in your Discussion (conclusion!)
Unlike Daphnia, this expt. gives good opportunities for explanantion!
Hypothesis
Note that this experiment is particularly GOOD for hyothesis.
As the concentration of trypsin, a protease is increased, the time for the casein solution to clear will
……………… and the rate of the reaction will ……………….. This is because, as enzyme
concentration increases …………………………. Continue, with the fullest explanation you can
give. Use your notes and your book and draw a predicted graph. (key words – collision, enzyme,
substrate, enzyme-substrate complexes, active sites)
Materials and Methodologies
Note that this experiment is particularly GOOD for PLANNING because we changed the
SNAB suggested method.
Write out your method and pay attention to detail. Use future tense so that if the CPR paper asks
for a plan, you can use this! Write this in bullet points for speed and simplicity. Eg (as a starter)

I will use a light sensor connected to a data logger, and a …. Watt light bulb. The light will
point into a reaction beaker of protease and milk, and the light sensor will be set up below,
as shown in this diagram/photo”

The light will be clamped at exactly ……… above the beaker, and the light meter will be
clamped ………… from beaker. If I were to vary either of these …….

I will carry out each ‘run’ as quickly as possible as I do not want ambient light to change
because …….
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I will measure ……….. ml of milk and add it to the beaker
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I will measure ……… ml of protease, having previously mixed the following concentrations
……………
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I will add the protease as I start the data logger and will record the light reading in Lux every
30 s for ……………s. I have carried out a trial that suggests that it is not possible to reach the
same end point with each concentration. Therefore a comparison of clarity (as shown by the
light reading) after a certain period of time is more appropriate

I will carry out as many repeats as time permits for each concentration of protease

I will repeat for each concentration of protease, namely ………..%, ………..%, ………..%,
………..%, ………..%, ………..%, & ………..%.
I will use 5ml of milk with 5ml of buffer solution, but NO protease, as a CONTROL
because………….
Note that this experiment is particularly GOOD for choosing a control, and that this is NOT
the same as ‘control of key variables’. Is it??
Control of key variables
Note that this experiment is particularly GOOD for control of key variables, especially pH
and temperature
In addition to keeping the light from the bulb constant and the position of the light meter constant in
relation to the reactants, as stated above,

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I shall keep the volume of protease at a constant ….. because ………….. (what would
happen)
I shall keep the volume of milk at a constant ……….. because …………….(what would
happen) and
I shall use milk that is of constant concentration (4%). If I were to change concentration of
the milk …………(what would happen)
(key words – again - collision, enzyme, substrate, enzyme-substrate complexes, active sites)

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I shall control pH by using buffered distilled water (at pH8, the optimum for trypsin) when I
dilute the protease. This is vital because any alteration of pH will affect the experiment (add
detail about rate of reaction/pH . Include the graph from last lesson or from the text book).
I shall attempt to control temperature by standing my prepared beakers of milk in a water
bath set at 32oC. This is close to the thermal optimum for trypsin and I have found (by trials)
that this is the temperature to which the light bulb heats the two solutions. Thus I will be
maintaining a constant temperature. This is important because (add detail about rate of
reaction/temperature. Include the graph from last lesson or from the text book)
There are great opportunities for the next bit in this expt. Make sure you read SNAB PRAC
SUPPORT 2 again, and my comments on your beetroot.

Precision means ………………………….. I will obtain precision by …
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Accuracy means ……………………………I will obtain accuracy by …
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Reliability means …………………………. I will try to obtain reliability by …
Important: say (in the context of the above) that a stop clock will let you measure to an accuracy of
=/- ?, a light sensor to +/- ……….; a thermometer to +/- ………a graduated pipette, to +/…………..
The choice of a small beaker is the result of trial and error: it gives great results. Try to think why it
might be better than a boiling tube or large beaker. Why is it best set up like this rather than with
the light to the side?
Risk assessment:
Note that this experiment is particularly GOOD for risk.

I am working with an enzyme therefore ………… (read and copy the risk assessment from
the yellow card)

I am working with a bulb adjacent to a liquid so …
Independent variable= the concentration of the protease solution.
Dependent variable= the time it takes for the milk solution to turn clear.
Results:
Important note
Analysis of my sets of results showed that it was not possible to obtain a common end point. It was
also difficult to choose an intermediate point in time at which point the lux values could all be
compared: the higher concentrations ran to completion very quickly indeed (within 180s) and the
lower concentrations took upwards of 20 minutes. When the reactions DID complete, end points
were quite different. For this reason, another set of results was taken and different protease
concentrations were used; 0.5%, ………. ………….. (continue). Using these concentrations it was
possible to compare the light values for each protease concentration at one point in time (120s)

I have chosen to display these results in a table/ spreadsheet because (copy from my notes
on beetroot or from SNAB Prac Supp notes)

I have put chosen to display the values for time (say where), and the values for the DV
(light in lux ……………… (say where) because ……………..T values for the IV (protease
concentration) I have put ……….… (say where)
Graphs

I have chosen to draw a line graph of protease concentration as a % (IV) against light levels
in lux, (DV) after 120 secs. This graph shows the relationship between the dependent and
independent variables. A line graph is appropriate because ……….. (copy what I said
about beetroot!!) It is appropriate to join the dots rather than draw a LOBF because …….

I have also drawn a line graph for one particular concentration to show the time of reaction
by graphing the change in light levels (lux) through time to completion. This allows me to
show that I worked with precision because …………………. . I can assume that these
results are accurate – ie …………..(define accuracy) because …………………
If you have graphed from Excel state the advantage of doing so.
Discussion
My first graph shows that the higher the concentration the ……….. light that is detected by the light
sensor as the protease hydrolyses the milk solution
Give as complete an account as possible.
Repeat, hopefully modified in some way, your hypothesis and rationale.
Note that this experiment is particularly GOOD for discussion (explanation of results.
Evaluation:
Ideas! NOT a fully inclusive list!

Would it have been better to stop at a constant end point (in lux). Why do you think this was
impossible?
Was it possible to eliminate or control ambient light?

Was there a heating effect from the bulb?. How would you control this?

What were the sources of random error? (define this) Hint: this was why a constant end
point was so difficult.

Was there a source of systematic error?

Finally (it’s appropriate to reflect on this in the evaluation). ARE your results accurate? Are
they RELAIBLE? Do you think they are VALID? Again, define these words from PRAC
SUPP SHEET 2, to help clarify what you write.

Could you suggest a real improvement in the method to counteract the various faults in the
experiment.
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