Enzyme Catalase
The enzyme catalase speeds up the decomposition of Hydrogen Peroxide into
water
and oxygen as shown here, 2H2O2-------------------*2H2O+O2. It is one
of the
fastest known enzymes and its turnover number is 6 million, which
means the
number of substrate molecules which one molecule of the enzyme
turns to products
per minute. This can be demonstrated by putting a piece of
liver into a beaker
of Hydrogen Peroxide, the fizzing shows a demonstration
of the enzyme in action.
AIM My aim is to examine how the concentration
of the substrate hydrogen
peroxide affects the enzyme catalase. INVESTIGATION
I am going to investigate
the effect of varying the substrate concentration
on enzyme catalase. I am going
to use 8 different concentrations and record
the time taken to collect 20ml of
gas in the gas syringe. I will repeat all
the 8 concentrations twice so I can
see if they match, spot out any anonymous
results and also I can work out the
average time it takes to produce 20ml of
gas at the certain concentrations. I
will vary the concentrations by
increasing and decreasing the amounts of
Hydrogen Peroxide and water.
PLAN First of all I will ensure I have enough
enzyme solution for the whole
experiments so the enzyme solution is standardised.
With the results I
get I will try to work out the ½ Vmax. I will do this
experiment at room
temperature so the enzymes get enough kinetic energy to
collide. I will need
80ml of the enzyme solution because I will use 5ml for all
of the experiment
and I will do 8 different concentrations and I will repeat
this
concentrations twice so that is 5x8x2= 80. First of all I will set out
the
equipment as I will show in the diagram then I will cut some pieces of
liver,
which is the source of the enzyme. Then I will grind the pieces of
liver with
the mortar and pestle, which will have sand and Di ionised water
(which is water
with no H ions in it its PH is neutral). The sand will help
cut open the cells
of the liver. I will take a funnel with glass wool in it,
I chose glass wool
rather than filter paper because the catalase could have
been adsorbed by the
filter paper. Then I will add 5ml of the enzyme catalase
to the conical flask
and for the substrate concentration of 10% I will add
2ml of Hydrogen Peroxide
and 18ml of water (18+2= 20, I will always use 20ml)
every time I when I will
increase the concentration by 10% I will increase
the H2O2 by 2ml and decrease
the H2O by 2ml. I will time how long it takes to
produce 20ml of gas in the gas
syringe. I chose the gas syringe rather than
to count the bubbles produced in a
measuring cylinder because it is easier to
use, the results will be more
accurate and the gas syringe reduces the
possibility of gas escape. I will
tabulate my results and highlight them in
some way so they are visible I will
interpret my results in to a line graph.
I will also added a line of best fit to
the results on the graph and with the
results I get I will work out the ½ Vmax.
Here is a blank copy of my
results table, which I fill in later when I get my
results. FAIR TEST To make
my experiment a fair test I need to ensure that all
the variables must be
kept the same for all the experiments except for the
concentration of
Hydrogen Peroxide. I will accurately measure out the Hydrogen
Peroxide
and enzyme solution using a pipette and measuring cylinder. I will use
glass
wool rather than filter paper because if I use filter paper then the
catalase
could be adsorbed by the filter paper, which will no longer make
my
experiment a fair test. I will time how long it takes to produce 20ml of
gas by
using a stopwatch accurately. For each concentration I will make sure
that there
is no excess catalase or substrate in the measuring cylinders I
use by cleaning
them. I will hold the rubber bung connecting the conical
flask and the gas
syringe so it does not open and let out any gas. PREDICTION
I predict that the
more the concentration of substrate the faster it will be
to produce 20ml of
oxygen if you increase the concentration there will be a
higher chance of
collision between the particles, but there will come a point
where all the
active sites are full and the rate should go constant. Enzymes
such as catalase
are protein molecules. They are used to speed up specific
reactions in the
cells. They are all very specific as each enzyme just
performs one particular
reaction. Once the amount of substrate molecules
added exceeds the number of
active sites available then the rate of reaction
will no longer go up. The graph
should look like this, I know this from
background scientific knowledge, from my
notes and textbooks. SAFETY ASPECTS
The safety precautions that I will consider
taking are that I am going to
ensure that I wear goggles because Hydrogen is a
strong oxidising agent and
if it gets into my eyes it could be irritating and
eat away at my cornea,
corneal burns can occur rapidly. I will also make sure
that the Hydrogen
Peroxide does not come in contact with my clothes or hands so
I will wear
an apron and gloves if the Hydrogen Peroxide does come in contact
with my
hands, which will cause whitening of the skin and stinging. I
will
immediately wash my hands thoroughly with water. Hydrogen Peroxide is a
strong
deodorizing and bleaching agent. It has a characteristic pungent odor.
If anyhow
I swallow the Hydrogen Peroxide I will drink water straight
away to dilute and
immediately contact a physician. If I break any apparatus
I will inform the
teacher straight away and I will clean the broken
apparatus. APPARATUS LIST
? Scalpel and chopping board ? Pipette ? 4
measuring
cylinders, (3) 100ml (1) 10ml ? Conical flask ? Gas syringe
?
Mortar and pestle ? Glass wool ? Rubber bung with
delivery tube ? Retort
stand with clamp and boss ? Stopwatch
? Sand ? Spatula ? Hydrogen Peroxide ?
Di ionised
water ? Source of enzyme (liver) I used these apparatus because it
was
the equipment available and suitable for my experiment. I used the gas
syringe
rather than to count the number bubbles produced in a measuring
cylinder because
it is more easier and accurate and it also gives a less
possibility of gas
escape. I used the stopwatch because it is accurate to
measure the time rather
than counting the time yourself. I used the spatula
to pick up bits of the sand
and put in the mortar and pestle in which I was
grinding the pieces of liver. I
used the scalpel to cut the pieces of the
liver and I cut the pieces on the
chopping board so I don’t cut into the
table that I was working on. I used the
mortar and pestle to grind the pieces
of liver. I used the glass wool rather
than filter paper because the catalase
could be adsorbed by the filter paper,
which will no longer be a fair test.
METHOD First of all I ensured I had enough
enzyme solution for the whole
experiments so the enzyme solution was
standardised, so if incase I am in
between an experiment and the enzyme solution
finishes then if you make the
solution again you might not get the right
concentration, so the experiment
will not be a fair test. I used pipettes to get
the solutions and I
accurately measured the amounts by using measuring
cylinders. I needed 80ml
of the enzyme solution because I used 5ml for all of
the experiment and I
done 8 different concentrations and I repeated these
concentrations twice so
that is 5x8x2= 80. First of all I set up the equipment
as shown in the
diagram then I cut some pieces of liver, which was the source of
the enzyme.
Then I grinded the pieces of liver with the mortar and pestle, which
had sand
in it and Di ionised water (which is water with no H ions init its PH
is
neutral). The sand helps cut open the cells of the liver. I then took
a
funnel with glass wool in it, I chose glass wool rather than filter
paper
because the catalase could have been adsorbed by the filter paper. Then
I added
5ml of the enzyme catalase to the conical flask and for the
substrate
concentration of 10% I added 2ml of Hydrogen Peroxide and 18ml of
water (18+2=
20, I always used 20ml) every time I increased the
concentration by 10% I
increased the H2O2 by 2ml and decreased the H2O by
2ml. A problem did occur at
one point when I was doing my experiment for
TRIAL 1 for the substrate
concentration of 30%, it took a long time, much
longer to get to 20ml of gas
produced its time was no where near the previous
concentration it had no
pattern, so I stopped it and I repeated it again then
it was alright it took
normal time it was in pattern with the other
concentrations. Probably the reason
for the reaction at that particular
concentration to take that long when I did
it first might have been that the
enzyme must of deteriated fast or when I put
the enzyme in the conical flask
with the substrate and put the rubber bung on
top I might of took a little
while to time the reaction, but then I repeated it
again and it was alright.
I timed how long it took to produce 20ml of gas in the
gas syringe. I chose
the gas syringe rather than to count the bubbles produced
in a measuring
cylinder because it is easier to use, the results will be more
accurate and
the gas syringe reduces the possibility of gas escape. I tabulated
my results
and highlighted them in some way so that they were visible I
interpreted my
results in to a line graph. I also added a line of best fit to
the results on
the graph. OBTAINING I used substrate concentrations from
10%--80% I
varied the concentrations by increasing and decreasing the amounts
of
Hydrogen Peroxide and water for example for the substrate
concentration of 10% I
used 2ml of Hydrogen Peroxide and 18ml of water, for
the substrate concentration
of 50% I used 10ml of Hydrogen Peroxide and 10ml
of water, etc. I used pipettes
to get the solutions and I accurately measured
the amounts using measuring
cylinders. I used a gas syringe to collect the
volume of gas produced. I timed
how long it took to produce 20ml of oxygen
using a stopwatch. To work out the
average time taken (in seconds) to produce
20ml of oxygen at different
concentrations, I collected the time taken for
TRIAL 1 and TRIAL 2 in minutes
and seconds and then divided the results for
each concentration by 2 then the
final results was converted into seconds. I
tabulated the results, highlighted
them and interpreted them on to a graph
with a line of best fit on the graph.
Here are my results of the 8
different concentrations repeated twice and a graph
to interpret them.
CONCLUSION My results show that the increase of concentration
of the
substrate, which was Hydrogen Peroxide, increased the speed of the time
taken
to produce 20ml of oxygen. My results do not support my prediction.
My
results do not fully go with my prediction which was that the more
the
concentration of substrate the faster it will be to produce 20ml of
oxygen if
you increase the concentration there will be a higher chance of
collision
between the particles, my results show the explanation of the
theory of kinetic
energy, but there will come a point where all the active
sites are full and the
rate should go constant, but my results did not show
that. I knew that the
prediction was true because of scientific background
knowledge, from my notes
and from science textbooks. I could not work out the
½ Vmax because my results
are not reliable and there could be lots of
possibilities that effected my
results such as, I should have repeated the 8
different concentrations more than
twice, which will give me more results to
compare, the temperature in the room
could have changed by a degree or two
which could have altered my results, there
might have been the possibility of
gas escape between the rubber bung and the
gas syringe which was connected
via a delivery tube, there is a slight delay
between pouring the Hydrogen
Peroxide into the conical flask, adding the
catalase, putting the bung on and
starting the stopwatch, which could give a
different result. I should have
got someone else to start and stop the
stopwatch, which is not possible if no
one wants to do that. I may have measured
the concentrations inaccurately,
which could have altered my results. EVALUATION
Looking back at my
experiment, the changes I could have made to improve and
benefit it could
have been, that I could have repeated the 8 different
concentrations more
than twice, which will give me more results to compare BUT
there was a time
limitation. I could have used better apparatus which would have
made my
experiments more fair and efficient, but that depends on the
availability of
equipment and better apparatus are more expensive and larger and
which make
them harder to handle and less safe to work with because of size and
the
apparatus could be new to me and I could mishandle it. I think the
apparatus
I had was the probably the best available. I could have tested
different sources
of the enzyme catalase e.g. apple, peas, etc. But that is
time consuming, and
again there was a time limitation. The obtaining of the
enzyme was quite time
consuming, my enzyme source was pieces of liver which I
had to firstly cut using
a scalpel, grind with a mortar and pestle with some
sand and Di ionised water
which wasted time, probably there could have been
an easier way to obtain it,
which would have saved me time, may be to repeat
my experiment again. The
hydrolysis of milk fats by lipase Lipase is a
digestive enzyme produced in the
pancreas. It flows into the small intestine
where it breaks down fats to fatty
acids and glycerol. In this experiment you
will investigate the relative
concentrations of lipids in three types of
milk: ? skimmed ?
semi-skimmed ? full fat The concentration of the lipids in
the samples
can be assessed by adding lipase to the milk. Lipase is a
digestive enzyme
produced in the pancreas. It flows into the small intestine
where it is
responsible for the hydrolysis of triglycerides to fatty acids
and glycerol. It
is possible to follow the reaction by monitoring the pH of a
mixture of milk,
lipase and sodium carbonate. A milk with higher lipid
content should release a
greater number of fatty acids in a given time period
and therefore its pH should
drop quickest. Method 1. Place 10ml fresh milk in
a boiling tube (1) and add 5ml
of dilute (0.05M) sodium carbonate solution.
Place the boiling tube in the water
bath to warm to 40°C for 5mins. 2. Place
1ml of 5% lipase in a boiling tube (2)
and place in the water bath to warm to
40°C for 5mins. 3. Set up the
datalogging equipment as shown below. 4. Pour
the milk with carbonate solution
into the warmed enzyme . Shake the boiling
tube and return to water bath. 5.
Record the change in pH for 8 minutes.
6. Rinse the pH probe in the pH7 buffer
solution and repeat experiment with a
different milk sample. Bile salts are
steroids with detergent properties
which are used to emulsify lipids in
foodstuff passing through the intestine
to enable fat digestion and absorption
through the intestinal wall. They are
secreted from the liver stored in the gall
bladder and passed through the
bile duct into the intestine when food is passing
through. Biosynthesis
represents the major metabolic fate of cholesterol,
accounting for more than
half of the 800mg/day of cholesterol that the average
adult uses up in
metabolic processes. By comparison, steroid hormone
biosynthesis consumes
only about 50 mg of cholesterol per day. Much more that
400 mg of bile
salts is required and secreted into the intestine per day, and
this is
achieved by re-cycling the bile salts. Most of the bile salts secreted
into
the upper region of the small intestine are absorbed along with the
dietary
lipids that they emulsified at the lower end of the small intestine.
They are
separated from the dietary lipid and returned to the liver for
re-use.
Re-cycling thus enables 20-30g of bile salts to be secreted into
the small
intestine each day. The most abundant of the bile salts in humans
are cholate
and deoxycholate, and they are normally conjugated with either
glycine or
taurine to give glycocholate or taurocholate respectively. The
conjugation is
important in identifying the bile salt for re-cycling back to
the liver.