Trypsin Solution and Enzymes

Aim: To investigate the initial rate of activity on trypsin at varying

concentrations of an enzyme.
Theory:
The majority of metabolic reactions that occur in the body are controlled by
enzymes. The job of enzymes is to fulfil its role as a biological catalyst. These are
substances that speed a reaction without being used up. They are therefore able
to control the speed at which the reaction, otherwise known as the rate of
reaction. Enzymes are globular protein with a specific 3D shape. Enzymes work
on substances called substrate, after an enzyme- substrate complex forms; it is
broken down into product.
Structure of an enzyme
Every enzyme has an active site that acts at the catalyst. The active site is a
deep depression in the enzyme that enables a specific type or shape of substrate
with a complimentary shape to fit into the active site and then be broken down
into product. This helps enzymes maintain their specificity as the enzymesubstrate complex can only be formed by the joining of a specific enzyme or
substrate. When this happens this lowers the activation energy, (the energy
required to start a reaction).The enzyme under investigation, trypsin, is produced
in the pancreas and is a type of protease meaning has the function of breaking
down specific proteins into amino acids. This, along with many other enzymes,
allow the human digestive system to function efficiently.
Trypsin is an enzyme that can break down many proteases. The most common
that are broken down in the human body include a mix of arginine ethyl esters or
amides that act a substrate.
Lock and Key hypothesis of Trypsin:

1) The enzyme trypsin has a globular shape with an active site.

2) A substrate of trypsin e.g N-Benzoyl-L-arginine ethyl ester will form an enzyme
substrate complex.
3) The substrate is broken down in product.

However, enzymes only work at best at what is known as optimum conditions. An

enzyme to work at its best must be able to operate at an
a) Optimum temperature
b) Optimum pH
c) As well as this we can maintain the enzyme and substrate concentration in
order to ensure an efficient reaction occurs.
Substrate concentration refers to the number of substrate molecules present in a
given volume of solution. It is a known fact that as the substrate concentration
increases, the rate of reaction is also faster; this is due to the fact that there is
more substrate to be able to take part in the enzyme catalysed reaction,
meaning it is broken down quicker, as most free active sites become occupied.
Hypothesis:
I predict that as the concentration of the substrate doubles the rate of reaction
(i.e the time taken for the reaction to be complete) will half.
Caesin Suspension
To test this idea I will use 5 different concentrations of trypsin and add them to
casein suspension (commonly found in milk). Caesin suspension contains a
protease that can be broken down by the trypsin enzyme. The way I will know
that a reaction is complete is when the casein suspension undergoes a colour
change. As I want this point to be quite obvious, I will use milk as my casein
suspension, this way; all the experiments will start of a white colour and
gradually turn colourless as the trypsin is added.
Equipment List
Equipment
Trypsin Solution

(INDEPENDENT
VARIABLE)

Caesin Suspension (milk)

Distilled water

Amount
5 different
concentrations
Different
dilutions will be
made.

Approximately
20ml ( to be split
later for

How does it help to test hypothesis

We will use the following
concentrations:
0%-(just water)
0.2%
0.4%
0.6%
0.8%
1%
I am using this range of
concentrations that will enable me to
get valid and comparable results. To
prove that the enzyme is effective in
its role as a biological catalyst, the 0%
of trypsin will be used. (This
concentration is predicted to take a
longer time)
To act as substrate
To prepare the dilutions and hence
vary the concentration of trypsin.

Test tubes
Pipettes

dilutions)
6
6

Blank Card with cross on it

Stopwatch
(DEPENDENT VARIABLE)
Beakers

To help test hypothesis effectively

One to hold trypsin solutions and one

for water

To hold experiment
To accurately add any solutions i.e.
casein.
This will be placed behind each test
tube- this is to ensure maximum
accuracy f our results. Not only will we
have the colour change as an
indicator but The point at which the
cross can be see is also an indication
the reaction has finished

Risks
This is a relatively low risk experiment, however to ensure there are one or two
precautions we must take. Firstly, as trypsin is a digestive enzyme, it is worth
preventing spillages as if this were to come in contact with the eyes, it could be
very damaging. For this reason we can wear eye protection. Also, if spillages do
occur wearing an apron to prevent clothes being damaged, is one way to reduce
this risk. Moreover, we must be careful when handling glassware in this
experiment and make sure test tubes are left in their test tube holder to ensure
they do not roll of the table etc.

Method:
1) Prepare the equipment needed for the experiment (given above)
2) Prepare 6 concentrations on enzyme solutions.
The following dilutions should be prepared:

Volume of trypsin (ml)

Volume of water (ml)

Final Concentration (%)

0.8

0.6

0.4

0.2

3) Pipette 5cm of the protein suspension into test tube.

4) Pipette 5 cm^3 of the trypsin solution into test tube. Mix thoroughly and
immediately start the stopwatch.
5) Record the time taken for protein solution to clear.
6) Repeat steps 1,2 and 3 of experience for the range of different
concentrations given above .

Results Table

Time in Seconds (s)

Trypsin
Concentration
(%)

Mean Time in
Seconds

0.2

1225

1471

984 (TOO
LOW)

1348

0.4

723

872

563 (TOO
LOW)

797.5

0.6

364

512

385

420.3

0.8

225

258

320

267.6

209

239

154

200.6

Dealing with Anomalies

Anomalies are written in blue, I deemed these as anomalous readings as they did
not quite fit the pattern of results therefore, to ensure accuracy of results I didnt
include these values in the average times of each concentration.

NB:the first value I feel should be somewhere within the 1220-1550 bracket
the second value should be higher, within the 600-900 bracket

Conclusion based on evidence.

I can see very clearly that as the concentration of trypsin increases the time take
for the reaction to finish (i.e. the milk/casein solution to go from white to
colourless or see the black cross on the card) decreases. This tells us that the
two factors are inversely proportional. As one increases the other decreases. This
proves my hypothesis to be correct as I stated that as the trypsin concentration
doubles the rate of reaction will half. This is seems to be the case with
approximately every reading i.e. 1348/2 =674 approximately around 700 but not
exactly the case. However, the general trend is clear to see. The rate at which
the reaction took place decreased with increased substrate concentration. This is
further reinforced with the fact that scientifically, if there are more enzyme
active sites becoming occupied with the increasing concentration of trypsin, the
reaction is quicker. It is seen that as the substrate concentration doubled from
0% to 1% the overall reaction time overall was 6 times quicker, again proving
trypsin to be an effective biological catalyst.

Evaluation of Conclusion

Conclusions made from my evidence were that:

1) As the substrate concentration doubled from 0% to 1% the overall reaction
time overall was 6 times quicker.
2) As the concentration of trypsin increases the time take for the reaction to
finish (i.e. the milk/casein solution to go from white to colourless or see
the black cross on the card) decreases.
3) The results proves my hypothesis to be correct as I stated that as the
trypsin concentration doubles the rate of reaction will half.

1) I would say that this is a reliable statement to make as The concentration

was in essence doubled from 0% to 1% with 0.2 gaps in between. Which
lead to the time decreasing from a value of approximately 1400 to 200
1400/200=6 therefore reaction was 6 times quicker.
2) This is also reliable as this is the general trend shown in the graph. 200.6
seconds is quicker then 267.6 seconds
3) Not exactly half as some values are either too high or too low to be of the
halfway point.

This shows my conclusion to be pretty reliable

Evaluation of method:
Overall I feel my method was very reliable I used certain utensils for example,
pipettes to ensure accuracy of the experiment. Moreover, it was easy to follow
with simple steps. There was however many flaws in the experiment. I feel as
though we should have done a netter job at controlling other variables. We only
left the test tubes to react in a room where temperatures may vary at different
times. Temperature should be kept constant for this reason if I were to do this
experiment again I would leave the test tubes in a after bath at 35 degrees as
this is the optimum temperature not just for trypsin but many other enzymes in
the body. Moreover, there was room for human error when pressing the
stopwatch as someones perception of white to colourless may be different to
someone elses therefore the time may not be as accurate. Therefore, next time
we should have only one person in charge of the stopwatch.

Overall, to take this investigation further, I would have liked to taken the results
of even more concentrations of trypsin to be more precise i.e 2%,3% and so on,
to provide an even more valid conclusion or I would have looked at other factors
such as the effect on temperature. I probably would have wanted to take this
investigation further until substrate concentration could increase no more and
the solution became saturated
I would have probably tested more enzymes such as pepsin another digestive
enzyme to see whether the effects are similar and compare my results.