A Plan of an Experiment to See the Effect of Varying Concentrations of Hydrogen Peroxide on Yeast.

A Plan of an Experiment to See the Effect of Varying Concentrations of Hydrogen Peroxide on Yeast

I have been assigned the task of viewing the results of varying concentrations of hydrogen peroxide (H2O2) on yeast. I will do this by placing varying concentrations of H2O2 into a constant amount of yeast, then I will measure the amount of O2 produced (every 10 seconds) and plot a line graph for each concentration. Using the immediate gradient for the initial rate I will then use these amounts and plot another graph to show initial rate of reaction.

Enzymes work as follows: Active Site =

Enzyme Substrate Enzyme Substrate Enzyme Products

Complex

The enzyme joins with the substrate in an area called the active site ( ) this is where the enzyme works. At the active site the enzyme forms temporary bonds with the substrate these bonds hold the substrate in position while the enzyme works.

An enzyme lowers the activation energy of a reaction. This means that instead of needing a kick start i.e. some heat, the enzyme lowers the energy needed to start the reaction, so the reaction takes place there and then. This can be seen in the graph below:

Using my AS Biology Text Book and my existing scientific knowledge I will now make a well guided prediction. The general graph for the effect of substrate concentration on an enzyme is as follows:

This is a preview of the whole essay

This is a graph based on the initial rate of reaction for an experiment. This is found by measuring the gradient of each of the lines done prior (in a standard graph (time = X axis, volume of gas produced = Y axis)) read at the fastest point. This point can be read after 10seconds or after 30seconds dependant on the results. The initial rate graph is always in direct correlation i.e. the more H2O2 the more O2 produced. Therefore based on this I will now predict the results for this experiment. I predict that the quickest oxygen will be produced at the highest concentration of hydrogen peroxide and the slowest volume of gas produced at the lowest concentration of hydrogen peroxide. I believe this due to if there is more substrate then the enzyme will experience turnover. This is where all of the active site of the enzymes are full and therefore the substrate has to wait for an active site to become available and therefore the enzyme rate of reaction will reach a plateau where it is still reacting but at a constant rate.

For this experiment I will use the following equipment:

Apparatus

Test tube rack
12 X test tubes
2 X retort stands
2 X bosses
2 X clamps
Screw clip
A 1 litre beaker
Barrel of a 20cm3 syringe
Glass delivery tube
A 1cm3 syringe
Rubber tubing
Needle
Measuring cylinder
Stopwatch
Water bath

Chemicals

18 X 5cm3 yeast
3 X 10cm3 of 1.0moldm-3 H2O2 (consisting of 10cm3 H2O2)
3 X 10cm3 of 0.8moldm-3 H2O2 (consisting of 8cm3 H2O2 and 2cm3 H2O)
3 X 10cm3 of 0.6moldm-3 H2O2 (consisting of 6cm3 H2O2 and 4cm3 H2O)
3 X 10cm3 of 0.4moldm-3 H2O2 (consisting of 4cm3 H2O2 and 6cm3 H2O)
3 X 10cm3 of 0.2moldm-3 H2O2 (consisting of 2cm3 H2O2 and 8cm3 H2O)
3 X 10cm3 of 0.0moldm-3 H2O2 (consisting of 10cm3 H2O)

I will set the above equipment up as follows:

The varying factors I will have to either control or investigate are:

temperature
pH
enzyme concentration
substrate concentration
inhibitors

I will now say how I will control or investigate the above variables. Temperature will be controlled by heating the yeast in a water bath. I will only remove the yeast from the water bath seconds prior to inserting the hydrogen peroxide. This will reduce the heat loss keeping the temperature constant.

pH will be controlled by the addition of a buffer. This will keep the pH at a constant amount.

Enzyme Concentration this will be kept constant by me adding the same amount of yeast each time. This will be 5cm3. This will keep the enzyme concentrations correct and therefore constant.

The variable which I am investigating is Substrate Concentration I will be measuring the effects of this by using 6 concentrations, 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0moldm-3.

Enzyme Inhibitors are substances which slow down the enzyme process. Competitive inhibitors attach themselves to the active site of the enzyme. These can stay there forever (irreversible) or they can leave (reversible). Uncompetitive inhibitors occupy other parts of the enzymes distorting the shape of the enzyme and therefore the active site distorts rendering the enzyme either permanently unusable (irreversible) or temporarily unusable (reversible). They are pretty hard to stop but unless I add an unknown substance there shouldn’t be any inhibitors.

In this experiment I will use 6 concentrations:

0.0moldm-3
0.2moldm-3
0.4moldm-3
0.6moldm-3
0.8moldm-3
1.0moldm-3

I will create the above concentrations using serial dilution, the following table is the amounts used to create each concentration:

I will be taking the readings 3 times each. I will fill the results in the following table:

Placing the average of the three results in the average row I will be able to see the results easily.

Prior to the writing of my method I have done some preliminary work. I did an experiment to find the effect of enzyme concentration on an enzymes rate of reaction. The experiment used the same equipment as I am going to use now therefore enabling me able to comment on the efficiency of the equipment and the method prior to carrying out the experiment. I believe there will be quite a few sources of error but they will be covered in the evaluation section. Overall the equipment was of a high standard of operation. Being easy to handle and producing relatively efficient results.

Based on the previous experiment I have done I will now state in detail the method I will be using to carry out.

First of all I will set up my apparatus as shown on the 2nd page. I will serial dilute all 6 of the concentration with enough to do 3 experiments each. Next I will get roughly 90cm3 of yeast and place it in a water bath (to get to a constant temperature). When the yeast is in the water bath I will add buffer (to keep the pH constant). I will then place 1cm3 of hydrogen peroxide into a needled syringe. I will then place 5cm3 of yeast into the clamp and place the bung firmly onto the tube. I will then insert the 1cm3 of H2O2 making sure all of the syringes contents are emptied I will start the stop watch. I will then take readings of the amount of O2 produced every 10 seconds. I will take a reading at 10, 20, 30, 40, 50 and 60seconds. I will then take a new test tube and place 5cm3 of yeast into it, place the bung on it, insert 1cm3 of the concentration used and time appropriately. I will repeat the above experiment 3 times per concentration for each of the 6 concentrations.

I will the plot the average of the results onto the first standard graph of concentration of H2O2 against O2 produced placing each line on the same graph. Next I will take the gradient for its initial rate of reaction, and plot a second graph.

I will take each concentration 3 times and then average it to obtain accurate results. Accurate results are a necessity for this experiment due to me plotting two graphs. This experiment will obtain accurate results by my very precise serial dilution of the H2O2 and the precise measuring of the amounts of yeast.

I will need to use specific safety techniques to make sure I am no risk of injury or death. I will follow the necessary precautions i.e. no running or always tie hair back etc… I will also need to check the hazcards on H2O2 to see if it is harmful or irritant. I will wear goggles and a lab coat at all times.

For this experiment I used the following resources:

OCR Advanced Sciences Biology 1

ME Class Notes

Daniel Rodgers - –