Key variable of my experiment: This is the main factor and it will be controlled into each inhibitor change in order to carry out the experiment successfully.
In relation to this another factor that will change will be water concentration to make my inhibitor into a solution, as iron reacts better in this state. However, this will be done proportionally to make 100% solution each time, which is equal to 10cm.
These two will be the only factors that can be varies as all other factors need to remain constant to produce a fair test with accurate and reliable results.
The inhibitor range that will be investigated- 10 8 6 4 2 0 , in proportion to the right amount of water. This reasonable but yet sufficient range was decided through my pilot study.
The range was kept limited due to time availability, if I had selected a larger range I may not be able to carry out repeats.
Factors that must remain constant throughout experiment:
Volume of Hydrogen Peroxide- This is the substrate in the experiment, it is used to test activity of inhibition. The experiment is dependent on the concentration of Hydrogen Peroxide because if there’s more substrate concentration, enzymes work slower than normal, whereas if there’s less substrate concentration enzymes work faster, which may vary my results making them not as reliable.
Catalase- This is the enzyme in the experiment; it is used to speed up reaction. The experiment is dependent on the concentration of Catalase because if I were to increase my enzyme concentration the rate of reaction would also increase. However, if I were to decrease my enzyme concentration, my rate of reaction would also decrease, giving me unreliable results. Therefore it would just be better to keep it at a constant concentration.
Buffer Solution (pH7)- This is the buffer solution in the experiment at pH7, which retains the pH level. If I were to change this pH level at different inhibitor range it would give me unreliable results, this is because any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters its shape, making the reaction faster or slower than it should be.
Pilot Study: I carried out a pilot experiment to determine whether or not there was any point in carrying out my experiment, if it was successful I would carry it out as my main experiment, if it wasn’t then I would’ve chosen to carry out another study.
It was also beneficial in other areas as it helped me develop my hypothesis along with other relevant research. It helped me to write an accurate account of my method as it showed me areas I needed to improve or add to. Finally it helped me take note of all the safety precautions and equipment that needed to be taken into consideration, this way I didn’t miss anything out.
My Pilot Experiment was quite similar to my main experiment. However, in my pilot study the experiment was carried out with 1g of lead nitrate in the form of powder, it wasn’t in solution. After doing a bit of research I thought if I did my main experiment with lead nitrate in solution I would get better results as Lead works better in solution.
Apparatus: 1 Medium Potato (enough for 24ml)- source of enzyme catalase
120cm pH7 buffer solution- to keep conditions constant
30cm Lead nitrate- source of inhibitor
30 cm Distilled water-
6 Burettes- to hold contents when reaction takes place
6 Clamps- to hold the burette upright and steady
1 Mixer- to mash potato and release enzyme catalase
1 Scalpel- to peel potato before putting into mixer
1 Digital stop clock- to indicate when to take readings
1 Filter funnel- to add contents into burette
2 syringes- to measure distilled water, catalase and lead nitrate
1 Black Marker- to record froth level on the burette
1 Pair of safety goggles- for safety when pouring H O into burette
1 Measuring cylinder-to measure contents
1 Weighing scale- to measure Lead Nitrate
1 Filter paper- to put the lead nitrate on while weighing
Method: To test out the concentration of inhibitor affects the rate of reaction, prepare the different concentration of inhibitor (Lead nitrate) in proportion to distilled water.
To prepare the Lead Nitrate solution all you need to do is place a piece of filter paper on the weighing scales and set the scale at zero. Then using a spatula pour 1g of lead nitrate onto the filter paper and carefully pour into 100cm of distilled water- stir until the powder has disappeared.
Then set apparatus as shown below, labelling each burette from 1-6 so u know the amount of lead nitrate solution to put into each burette in proportion to distilled water.
- Using a scalpel peel the skin of the potato. Chop into small chunks and place into the mixer. Let the potato crush in the mixer for atleast 45 seconds until plump and liquidy smooth. We do this to provide us with the enzyme catalase for the experiment.
- Once this is done separate 4ml of the crushed potato into burette 1, (leaving the rest if the potato for the other burettes) using a syringe. We use a syringe to do this as it is easy to measure with and is accurate.
- Next add 20cm of pH7 buffer solution with a different syringe. This is added to keep conditions constant during the experiment.
- Then add the pre-made lead nitrate solution and add the amount required into burette 1 as shown on the table above (i.e. burette 1 would have 10cm distilled water and 0 lead nitrate solution).
- Finally put your safety goggles on, and add 2cm of hydrogen peroxide (H O ).
- To ensure everything is mixed in properly for the experiment to occur successfully remove the burette from the wooden clamp stand, place your thumb at the top end so the contents don’t spill out and tip upside down two or three times so the contents are shuffled properly.
- After the burette is safely placed back in to the wooden clamp start the stop clock and take readings of the increase in froth level every 30 seconds for 10 minutes.
- Repeat steps 2-7 for burettes 2-6. Concentration of lead nitrate will differ for each burette, check on table to see what proportion of distilled water to add to each lead nitrate concentration in each burette.
Results:
10cm Distilled Water
0cm Lead Nitrate Solution
8cm of Distilled Water
2cm of Lead Nitrate Solution
6cm of Distilled Water
4cm of Lead Nitrate Solution
4cm of Distilled Water
6cm of Lead Nitrate Solution
2cm of Distilled Water
8cm of Lead Nitrate Solution
0cm of Distilled Water
10cm of Lead Nitrate Solution
ANALYSIS: After summarising my results into a table and plotting it into graph paper as a line graph, I can see that there is some degree of a basic trend represented.
On graph 1 where the result is dependent upon the increase in froth level, it shows that there was relatively little activity being carried out when there was a high concentration of lead nitrate. However as the lead nitrate concentration decreased there was relatively more activity going on, this is because there was a high increase in froth level.
In graph 2 the results obtained show that when lead nitrate concentration was between 0%-40% the volume of froth level was relatively high to when the concentration of lead nitrate was between 60%-100%- it had decreased by quite a significant amount.
Prior to the investigation I carried out some research. I found that lead nitrate is a heavy metal which acts as an inhibitor, as it binds to the enzyme and denatures it, and therefore slows down reactions.
This is expressed in my results- increasing concentrations of lead nitrate (inhibitor), has an effect on reaction, it slows it down.
After carrying out my experiment and obtaining all my results, I can see that there are a few questioning anomalous results, as can be seen by the two graphs.
In graph 1 the results obtained show that some of the results may have been unexpected, this is because I would’ve expected to seen a gradual decrease in rate of reaction as inhibitor concentration increased. I.e. completely dilute solution -all distilled water) would’ve had the most reaction as there were no traces of lead nitrate acting as an inhibitor to slow it down, and so would be at the top. As the solution became more and more concentrated with lead nitrate concentration the rate of reaction would decrease quite significantly as this is what my research suggested and would’ve therefore been supported by my hypothesis.
Instead my graph 1 shows me that all water reacted less than other solutions which contained more lead nitrate solution, this may have been due to an error in my method or preparation. In graph 2 my results were also quite varied. In this graph I expected my graph to slope downwards in a negative correlation. However, I got 2 major anomalies, one at 0% where I would’ve expected the volume of froth level to be higher than it was, because there was no lead nitrate solution present to act as an inhibitor. Another anomalous result occurred at 60%; here I also expected the result to be higher than it actually was.
However, I got a range of results which supported my hypothesis-because the burettes which contained low lead nitrate solution concentration generally had a faster rate of reaction than burettes which contained higher amount of lead nitrate solution concentration. Nevertheless, I expected more accurate results; the reason why I may not have achieved it may be due to my earlier assumption, where an error may have occurred in my method and preparation and caused these odd results.
From carrying out this investigation, I can see that inhibitors have some degree of effect on rate of reaction- the addition of an inhibitor prevents the normal enzyme- substrate complex being formed. The reaction rate is reduced.
The kind of inhibitor I believe lead nitrate is, is non- competitive, because when I carried out my pilot study I increased the amount of substrate concentration to see if it had an effect. I did this because according to research, if the inhibitor is non- competitive increasing substrate concentration will not reduce the effect of the inhibitor. Whereas, if it were competitive, increasing substrate concentration will reduce inhibition. This is because, as the substrate and inhibitor are in direct competition, the greater proportion of substrate molecule the grater their chance of finding the active sits, leaving fewer to be occupied by the inhibitor.
Evaluation: During my investigation there were many limitations that could have affected my results, and the way the conclusion was shaped.
A major limitation was TIME, due to a limited amount of time I wasn’t able to carry out replicas. Also only 6 different concentrations of inhibition could be tested. This means only a very general, overall trend can be identified across the results. Patterns between these values can only be approximated and are not necessarily very reliable or accurate.
Another limitation may have been that I may have used different amount of surface area of potato-due to human error, because when measuring 4ml of catalase into the syringe, there may have been an air bubble, which may have given me less than 4ml of catalase. This can be said about the substrate concentration also- decreasing substrate concentration can slow down rate of reaction. This was used to maintain the pH level in the experiment. However
During the investigation I also had to be aware of precautions such as p H buffer solution. This was used to maintain the pH level in the experiment. However if I had varied the pH level, it may have resulted in an increase or decrease in rate of reaction. This is because pH is a measure of hydrogen ion concentration. By breaking the hydrogen bonds which give enzyme molecules their shape, any change in pH can effectively denature enzymes. Each enzyme works best at a particular pH- (7 being a neutral point), and deviations from this optimum may result in denaturation.
Another precaution was taken with safety equipment. As I was handling hydrogen peroxide, the safety precaution I had to take was to wear safety goggles, especially when pouring it into the burette.
To further my investigation, certain experiments could be carried out into the effect of inhibitor on rate of reaction.
The major one being to carry out repeats at each inhibitor change, this will enable me to get more reliable and more accurate results, as I will be able to work out he average of this and be able to draw a graph of error bars, which may portray my present obtained results in a different but precise way.
I could also test at even more narrower inhibitor changes, to give me more accurate results.
Perhaps I could use also try and use a different source to provide me with the enzyme catalase, this is because the desired amount of catalase from the potato may be less or more than what I want it to be. I t was quite hard to measure and there wasn’t really a quick way of knowing- making my results negotiable. Consequently, maybe using catalase from yeast would’ve been easier to use and handle. Specific amounts could be measured much more precisely, and may give more precise results.
Another way could also be to use a gas syringe instead of a burette, as this would increase time availability and be easier to record the froth level, because it saves time calculating the froth level increase and gives exact readings. Answers won’t need to be rounded by the calculator.