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The Effect Of Enzyme Concentration On Enzyme Activity

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The Effect Of Enzyme Concentration On Enzyme Activity

The pancreatic duct in individuals who have cystic fibrosis frequently
becomes blocked, reducing or preventing the release of pancreatic
enzymes into the small intestine. The aim of this activity is to
investigate the effect of a reduction in enzyme concentration on the
rate of reaction, in this case the breakdown of protein by protease
enzymes.

Aim – Milk powder contains a white protein called casein. A white
suspension of milk powder clears on the addition of the enzyme
trypsin. The aim of this experiment is to investigate the effect of
enzyme concentration on enzyme activity. We will observe how fast the
milk turns transparent by changing the concentration of the enzyme on
milk.

Hypothesis – The higher the concentration of enzymes, the faster the
protein will be broken down, and therefore the egg white will turn
transparent faster.

Appropriate observations have to be made to validly test a hypothesis
or idea. The term “observations” is taken to include measurements.
Validity is the measure of confidence that can be placed in a
conclusion. After deciding upon a dependant variable to vary within
the experiment, the method of measurement should also be known, the
measurement taken should be valid e.g. the volume of oxygen collected
in 30 seconds is a valid measurement of rate of catalase activity.

Valid results are derived through precise, repeatable measurements and
observations, made with apparatus and experimental procedures that are
suitable for the task.

Ideally a large number of replicates (repeat measurements) should be
taken, and any results that vary considerably from the others should
be repeated or discounted. A mean can be calculated to be
representative of the set of results. The pressure of time usually
puts a limit on the number of replicates that can be taken.

To be reliable, measurements should also be accurate. To be accurate a
measurement should be close to the true value. Precision involves the
choice of apparatus and the skill with which it is used.

I had to also be aware of the different types of error that could
occur in my experiment, systematic, random, and human errors. To
overcome systematic problems I will need to know how to use apparatus
correctly, to avoid human error I will have to give my full attention
and concentration towards the experiment. To avoid random errors, I
have to control changes in the material used or the conditions in
which they are carried out in, as this will all affect the final
result we produce.

If we follow this we should be able to produce valid results.

Apparatus –

To carry out this experiment, the following apparatus are needed:

· Standard alkali trypsin solution

· Milk powder

· Standard laboratory glassware and apparatus

· Stop clock

· Ruler

· Thermometer

Safety – An important aspect of good practical work is safety. To work
safely during practical work, I made sure I followed each of the
following rules.

1. Do not bring non-essential materials into the lab. Keep bags and
coats outside of the lab as it could get in the way, and cause
injury.

2. No food or drink is allowed in the lab at any time. We will be
working with some fairly nasty chemicals and some exercises
involve bacteria. It can be dangerous if the chemicals r ingested.

3. The eyes are particularly sensitive to chemicals, and provide a
good portal of entry for microorganisms; the best way to avoid
contact is to keep your hands away from your face as much as
possible and to wear safety goggles at all times. Gloves may also
have to be worn when handling hazardous chemicals.

4. We will be working with stains in some exercises that will not
wash out of cloth, and so lab coats are needed.

5. Keep long hair tied back and ties tucked in, as we will have open
flames in some exercises.

6. Dispose of waste material properly. There are specific, clearly
labeled containers for glass and contaminated materials, they have
to be disposed of correctly otherwise this could cause problems
later on. We are also required to maintain a clear surface
worktop.

7. Alert the instructor to any spills or breakage. These accidents
need to handled correctly; we are not authorized to clean it up
ourselves.



Variables – The independent variable is the factor that is being
varied. A rang of values for the independent variable can be chosen.
The dependant variable depends on the value of the independent
variable. The dependant variable is the one that is measured in some
way. Any other variables that may affect the dependant variable should
be controlled (kept constant) in order to produce results that are
valid and reliable.
======================================================================



The independent variable within this experiment is the concentration
of the trypsin. The dependent variable within this experiment is the
time taken for the milk to digest. The constant/control variables of
this experiment are the substrate (milk) concentration, the
temperature and the pH.
=====================================================================

To keep the temperature constant in this experiment, we could have put
the test tubes in a water bath, and set the temperature to 25°C, so
that it would not affect the overall results. We did not do this, as
we discovered, from the preliminary trial, that the experiment only
lasted about five minutes maximum, and so temperature would not change
dramatically in such a short space of time. We did however, close all
windows and switch of radiators, we kept the experiment going in one
area.

This variable could have affected the end results if it had not bee
controlled. If there were dramatic changes in room temperature,
results would not have been reliable. As temperature gets higher,
enzyme activity increases – enzymes work at its optimum at about 37°C,
if temperatures get too high, they become denatured and no longer
work. As temperatures decrease, enzymes become less active and stop
working if temperatures are too low. So it is important to keep the
temperature constant in order to see the effects of enzyme
concentration on enzyme activity.



Method –First, I will take the pipette and put the solution in the
beaker of water and then use the stopwatch to time how long it takes
to turn clear. I will know when it turns clear, as I will be able to
see through it and read the writing. I will record the time it takes
to turn transparent, in a results table.
====================================================================

Enzyme Concentration

Volume of 1% trypsin

(cm3)

Volume of water

(cm3)

0

0

5

0.2

1

4

0.4

2

3

0.6

3

2

0.8

4

1

1.0

5

0

A solution was made that meant that we could judge what the outcomes
were compared to that. This was made up of 5cm3 of milk, 5cm3 of
distilled water and no trypsin. This as I expected showed no
transparency, which means that then the enzyme, actually works. Once
all the solutions were prepared, 5cm3 of enzyme was added into a
solution; by repeating a few more times I was able to get the next few
results.



Results –
=========

Enzyme concentration

1 - Time taken for milk to Digest (min/sec)

2 - Time taken for milk to Digest (min/sec)

0

-

-

0.2

4:43

4:13

0.4

2:33

2:51

0.6

1:56

1:14

0.8

1:34

0:59

1.0

1:12

0:51



The results show that as the enzyme concentration increases, the time
taken for the milk to digest decreases. Therefore the time taken for
the protein to be broken down by the enzyme decreases, as the
concentration of enzymes increased.
=====================================================================



Evaluation –
============



When I look at my results I can see that the increase in concentration
actually has a faster reaction of the enzyme and the casein so the
more of this enzyme there is the faster this can be broken down and so
the rate of digestion is directly proportional to the amount of
concentration of enzyme there is. I.e. the higher the concentration
the faster the reaction and then the lower the concentration the
slower the reactions are. In this case I can say that my hypothesis
was right as I predicted that this would happen.
======================================================================

When looking at any of the results in biology we have to see that they
are reliable and accurate. Reliability is when we take a look at the
possible errors of the experimenter and see if he stopped the stop
watch in time. Also we have to look at the accuracy e.g. if the
glassware has got a big percentage error. If so how we need to reduce
them. Also in the stopwatch as it can only go to seconds perhaps it
would be better if we used milli seconds. I believe that the
reliability of the results is low in this case when we look at all the
possible errors. We could only take the person who did the experiments
word in that the point in which he stopped the watch was the exact
point that he could see the writing on the other side. A person’s
judgement may be different to another person and therefore we will
never be sure because of the accuracy of the human eye. For example
one may have recorded the time when the solution was misty however
another person may have recorded the time when the solution was
completely clear.

Although the results were good, I could see a few anomalous results
that may not be that accurate. I can identify that there is a
significant difference between some of the results, for example the
0.8 and 0.2 enzyme solution. The variance of this suggests that there
is an error within the results. The reason for this error may be
identified as error systematic error and random error. An example of
systematic error is if the solution was made up incorrectly. For
example too much enzyme may have been added and too little water, if
the solutions were made up incorrectly than the true result is not
correct at anytime.

Random error can also been the reason for the error within the result.
An example of this is tiredness of the determining the end point of
the solution. Also, as time was limited many of the recorded results
may have been rushed. As a result of this, the accuracy of the
results may have been affected. Like I said before the end point of
each solution may not be correct it may always be different.

To gain a more reliable result, more repeats should have been done and
an overall average should have been worked out. Also other variable
such as the concentration of the substrate i.e. the milk is to be kept
constant and the temperature is also to be kept constant.

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