Enzyme Concentration: If there is insufficient enzyme present, the reaction will not proceed as fast as it otherwise would because there are not enough enzymes for all of the reactant molecules. As the amount of enzyme is increased, the rate of reaction increases. If there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate. Reaction rate therefore increases as enzyme concentration increases but then it levels off. For my experiment, my enzyme concentration is going to be constant.
pH Concentration: pH is a measure of the acidity or hydrogen ion concentration of a solution. It is measured on a scale of 0-14 with pH values below 7 being acidic, values above 7 being basic and a value around 7 is neutral. As the pH drops into the acidic range an enzyme tends to gain hydrogen ions from the solution. As the pH moves into the basic range the enzyme tends to lose hydrogen ions to the solution. In both cases the changes produced in the chemical bonds of the enzyme molecule result in a change in conformation that decreases enzyme activity.
Hydrogen ions interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement. A pH which is very different from the optimum pH can cause the denaturalising of an enzyme. When you add the sulfuric acid to your Catalase reactions you lower the pH below the range where Catalase is functional, and the reaction stops. In the case of Catalase, the optimum pH is approximately pH 7.0. That is, Catalase works best at a neutral pH. If the solution is too acidic (low pH value) or too basic (high pH value) the Catalase is inactive - no longer functions as an enzyme, but a few enzymes can work at extreme pH, such as protease enzymes in animal stomachs, which have an optimum of pH 1. My experiment will be kept at a pH of around 7 (which is fairly neutral).
Salt Concentration: Every enzyme has an optimal salt concentration in which it can catalyze reactions. Too high or too low a salt concentration will denature the enzyme. The salt concentration of my experiment is going to be moderate.
Presence of Activators: A molecule that interacts with an enzyme and increases its activity is an activator. This is going to be considered in my experiment.
As we have learnt above that the active site of an enzyme fits one particular substrate perfectly, it is possible however for some other molecule to bind to an enzyme’s active site if it is very similar to the enzyme’s substrate. This could inhibit the enzyme’s function. If an inhibitor molecule (an inhibitor is a molecule that interacts with the enzyme and decrease its activity) binds only briefly to the active site then there is competition between it and the substrate for the site, but if there is much more of the substrate than the inhibitor present, the substrate molecules can easily bind to the active site in the usual way and so the enzyme’s function is unaffected.
However, if the concentration of the inhibitor rises or the substrate falls, it becomes less and less likely that the substrate will collide with an empty site and the enzyme’s function is inhibited. This is what we call Competitive Inhibition. It is said to be reversible because it can be reversed by increasing the concentration of the substrate. Sometimes, the inhibitor can remain permanently bonded with the active site and therefore cause a permanent obstacle for the substrate. Here no competition occurs as it does not matter how much substrate is present, so this is non-competitive irreversible inhibition.
A different type of inhibition takes place if a molecule can bind to another part of the enzyme (allosteric site) rather than the true active site. This can seriously disrupt the normal arrangement if hydrogen bonds and hydrophilic interactions holding the enzyme molecule in its 3D shape. In this effect, ripples spreads across the molecule to the active site making it unsuitable for the substrate to bind with. The enzyme’s function is blocked no matter how much substrate is present so this is another type of non competitive inhibition. This condition can be reversible or irreversible, depending on whether the inhibitor bonds briefly or permanently with the enzyme. In the case of Catalase the active site is the haem group.
Below is diagram of a non competitive inhibition
Below is diagram of competitive inhibition
One important last thing I also learnt about inhibition of enzyme function is that even though it can be lethal, it is essential in many situations. For example, metabolic reactions must be very finely controlled and balanced, so no single enzyme can be allowed to “run wild” constantly churning out more and more product. One way of ensuring that this cannot happen is to use the end- product of a chain of reaction as an enzyme inhibitor.
Hypothesis: After all my background research, I predict that as temperature increases (with my hydrogen peroxide and Catalase concentration kept constant) the rate of reaction also increases. The rate at which Catalase will convert the hydrogen peroxide into oxygen and water will increase because increase in temperature increases the kinetic energy of the hydrogen peroxide molecules; therefore it will collide more successfully with the active site of the Catalase. I also predict the highest rate of reaction will be between temperatures of 40oC – 45oC (optimum temperature) and that at temperatures exceeding this, the rate of reaction begins to slow down, because the conformation of the 3D shape of the Catalase begins to change, therefore its activity also begins to slow down.
Below is my prediction graph:
My prediction graph shows that as temperature increases from 10oC – 40oC the rate of reaction also increases (maximum at 40oC), and rate of reaction will start to decrease after 40oC; this is an evidence that as temperature increases past the optimum temperature the enzyme starts to denature therefore its activity is also affected.
Variables
In this experiment I will not be controlling many variables as most of the factors are kept constant throughout the experiment. The only thing we will be changing is the temperature. Because the temperature I’ll be starting with for my actual experiment is more than room temperature, each time I want to increase the temperature I will place the conical flask or the beaker containing the solution in the water bath, with a thermometer inside in order to measure desired temperature.
I will keep constant key factors such as the amount of liver used; 0.3 grams of liver will be used. Also the substrate concentration will be kept the same. The amount of substrate to be used is 8ml. We know we cannot add more as it will cause results to vary. As for the liver we must keep the liver amount the same. As stated before, increasing surface area will increase the rate of reaction. So we have to keep the liver size as accurate as possible.
I carried out a number of different temperatures, starting at 35˚C and going up by 5˚C to 55˚C. For each temperature I repeated it three times.
PRELIMINARY METHOD
- Put on your lab coat.
- Put on your gloves and goggles.
- Collect all the necessary apparatus for the experiment.
- Label all the flask / beakers containing substance.
- Arrange apparatus
- Heat water to desired temperature and pour into the beaker (make sure it doesn’t enter conical flask).
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Measure 10cm3 of H2O2 with pipette and pour into the conical flask.
- Cut three masses of liver 0.8g, 0.2g, 0.3g.
- Place thermometer into conical flask and into beaker, to ensure they have the same temperature.
- Prepare stop watch.
- Place liver into the conical flask and immediately close the top of the conical flask with rubber stop from the syringe.
- As soon as that is done, start your stop watch and take readings on the syringe at desired time interval.
- Empty the conical flask and clean with water
- Push the glass rod inside the syringe back in.
- Repeat all this procedure but with different temperatures.
Apparatus
Rubber gloves
Safety glasses
Vaseline
Large beaker
Small beaker
Pipette
Syringe
Retort stand
Stopwatch
Hydrogen peroxide
Lamb liver
Knife
Tile
Tweezers
Conical flask
2 thermometers
Kettle
Electronic scale
BELOW ARE MY PRELIMINARY RESULTS FOR MEASURED TIME IN SECONDS AND VOLUME OF OXYGEN IN cm3
Volume of Hydrogen peroxide used: 10cm3
Mass of lamb liver: 0.8g
Volume of Hydrogen peroxide: 8cm3
Mass of lamb liver: 0.2
Volume of Hydrogen peroxide: 8cm3
Mass of lamb liver: 0.3g
The main aim of my preliminary experiment is to able to work out the right volume of hydrogen peroxide and the right mass of liver for my actual experiment. The temperatures I used for this preliminary experiment are 10oC, 20oC, 30oC, 40oC, 50oC and 60oC; I repeated each temperature three times. In order to be able to get the right quantity of my factors I kept temperature at normal optimum temperature of most enzymes (40oC); I started to vary volume of hydrogen peroxide and mass of liver, I took readings of oxygen displaced using a syringe for each volume of hydrogen peroxide and mass of liver I tried, I then compared the results so as to get the right values. Finally I went for 8cm3 of hydrogen peroxide and 0.3grams of liver because on my results table it looks most reasonable to me compared to the others.
In conclusion, the overall results gained from my preliminary experiment tell that enzyme and substrate concentration affects rate of reaction even at optimum temperature.
METHOD
- Make sure before you start the experiment that you are wearing your lab coat, gloves and goggle; this will prevent direct contact of the chemicals with your eyes, hands and your whole body.
- Collect all necessary apparatus for the syringe method and set up accordingly; I am using the syringe method over the inverted method basically because the inverted method is less accurate than the syringe method; I mean it is less accurate in the sense that bubbles of oxygen might lead to excess water being displaced from the cylinder and also if the oxygen is produced quite quickly it might be compressed by the water causing it to have a different level of pressure.
Inverted method
- Lubricate inside of syringe to reduce friction when oxygen is being displaced during experiment.
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Measure 8cm3 of Hydrogen peroxide using the pipette and pour into the conical flask. Make sure no air bubbles are introduced as this may affect the volume used, which will then affect the overall result.
- Cut out some liver and using the electronic scale measure 0.3grams.Make electronic scale reads zero before using it to scale.
- Label all the flask/beakers containing solutions in order to avoid confusion.
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Prepare 500cm3 of water with the beaker and heat alongside the conical flask containing the hydrogen peroxide in a water bath to desired temperature. Make sure thermometer is placed in both the beaker and conical flask in order to ensure they are both at same temperature.
- Prepare Stop watch
- Using the tweezers place liver into the conical flask containing the hydrogen peroxide and immediately close the conical flask with the rubber cork connected to the syringe. As soon as the liver drops into the conical flask start the stopwatch and take readings from the scale of the syringe at desired time interval e.g. every 5seconds.
- After all this is done push glass rod inside the syringe back in and empty all apparatus containing solution. Wash all apparatus with water.
- Repeat procedures but with different temperatures.
The temperatures I will be using for this experiment are 5oC,10oC,15oC,20oC,25oC,30oC,35,40oC,45oC,50oC and 55oC, each temperature will be repeated three times.
Diagram of experiment
Risk Assessment
- When using the Hydrogen Peroxide always make sure to have safety glasses on your eyes for protection. Hydrogen Peroxide is corrosive and irritant so safety glasses must be worn at all times of the experimental work. Also if the Hydrogen Peroxide is spilt on hands or on skin, make sure the affected areas are thoroughly washed with water. It is also best to have a laboratory apron or coat on during the experimental work. This is because Hydrogen Peroxide can damage garments you are wearing if it is accidentally spilt on them. Also use the thermometer to gently stir the water in the bath as it is being heated; this is so that the heat can be distributed out evenly throughout the water bath.
- Take care when temperatures of the water heated in the water bath are increased. If you decide to investigate on temperatures above 40˚C, extra care is needed to be taken with handling the water bath because it can get very hot. Therefore it is best to let the hot water bath cool down after your experiments have been completed before handling it again.
- Note that the Bunsen burner must be adjusted to the blue flame and heat proof mat should always be present underneath the burner. Also take care when using the burner and make sure you and your garments are away from the flame to stop any accidents from occurring.
- Put the glass wares in the middle of the table, to prevent them from dropping.
- Make sure you put labels to prevent any confusion between different chemicals.
- Wash equipment as any dirt could cause faults in the experiment.
Initial rate of reaction
Initial rate of reaction = 1/time (where 1 can be any number)
Here I will be taking the initial rate for 10s, for each temperature
Analysis
Looking at my results we can see that temperature does affect the rate of reaction in an enzyme catalyzed reaction. As temperature increases the rate of reaction also increases, this is because as temperature increases the kinetic energy of reacting particles also increases, thereby creating more successful collision, and more successful collision means increased rate of reaction.
From my main results table, it is obvious for every time interval that the rate of reaction gradually increased as temperature increases from 30oC- 40oC and was highest at 45oC, afterwards the rate of reaction started to decrease till the final temperature of 55oC. This is also buttressed by the results for the initial rate of reaction; the initial rate of reaction increased as temperature increases from 30oC – 40oC and was highest at 45oC, afterwards the initial rate of reaction decreased till the final temperature of 55oC.
Going on to my graph of initial rate of reaction against temperature I can see that the highest rate of reaction for the whole experiment was at 45oC, I can
therefore interpret that the optimum temperature of Catalase in the lamb liver used for this experiment is around 45oC.
There was an anomaly on my graph of initial rate of reaction against temperature; this was the optimum temperature of the Catalase in this reaction and was an anomaly because it was too high for optimum temperature; the fact that the liver was not at the same temperature as hydrogen peroxide raised the optimum temperature of the Catalase.
Conclusion
In conclusion my experimental work have supported my hypothesis that as temperature increases in an enzyme catalyzed reaction, the rate of reaction also increases up until the optimum temperature and after which rate of reaction starts to decrease. Also my experiment supported my background research that enzymes denature at temperatures higher than optimum temperature; looking at the results table the decrease in rate of reaction for temperatures higher than 45oC shows that the enzyme has started denaturing (changing shape).
Evaluation
I have to say my whole experiment was quite a success in the aim of finding out how the temperature of Catalase affects the breakdown of hydrogen peroxide, although there were some limitations and minor errors which must have contributed to some of the inaccuracies in my results. Most of the errors that contributed to the inaccuracies in my experiment came from the experimental procedures. One of limitations that I was faced with was the inability to keep the liver in the same condition through out the experiment, because the experiment took a few days to complete both the quality of the catalase and the equipment used would have changed; using liver samples from different conditions means that the livers will contain different amount of Catalase, also long term storage of the liver can results in deactivation of the catalyst and thus affecting the overall activity of the enzyme; this was the most significant limitation to this experiment as we don’t know the difference in concentration of Catalase in each liver sample used. The experimental techniques was quite suitable in the sense that I used the more accurate syringe method over the less accurate inverted method, this made the collection of displaced gas easy and also made taking of readings easier.
Time was also part of the limitations, as I would have loved to try a larger range of temperatures with small intervals, say like every 2oC. This will also provide a more accurate graph that shows a more accurate optimum temperature. Another limitation was that I wasn’t able to keep the Catalase and the hydrogen peroxide at the same temperature, Catalase used was at room temperature (25oC), these accounts for the reason why the enzyme was still working almost normally at 50oC and 55oC.
Also some of the main errors includes gas collection( placing the cork on top of the conical flask), as I wasn’t fast enough in placing the cork/bung on top of the conical flask, some oxygen must have been lost in the process which in turn added to some of the inaccuracies in the results, also the fact that not the same equipment was used through out the experiment must have caused some slight changes in the result which must have contributed to the anomalies in the results table; friction in the gas syringes vary, therefore using different gas syringe may affect the results. Also the fact that the experiment was done on different days was a problem, because then we had to use the liver sample kept in different conditions and from different source every time we wanted to the experiment.
The experimental evidence was quite reliable; this can be buttressed by looking at the closeness in most of the values in my results table even though there were some anomalies, for example at 40oC the volumes of oxygen displaced was 30cm3, 31cm3and 30.5cm3, we can see that this values are very close with a difference of 0.5cm3 between 31cm3 and 30.5cm3 and a difference of 1.0cm3 between 31cm3 and 30cm3; there was an anomaly on my graph of initial rate of reaction against temperature, at 40oC because it didn’t go with the curve; also there were some anomalies in my results table, for example at 40oC the volumes of oxygen displaced at 10seconds was 14.5cm3, 17cm3 and 17.5cm3, we can see that there was a clear difference between 14.5cm3 and the other two values, also another example was 35 oC the volumes of oxygen displaced at 45seconds was 73cm3,71cm3 and 65cm3, here also we can see that there was a clear difference between 65cm3 and the other two values; I think that reason for all this anomalies narrows down to the fact that the experiment was done on different days and condition which may have affected the amount of Catalase in the liver used and also the fact that the catalase and hydrogen peroxide were not kept at the same temperature, for instance the hydrogen peroxide used was heated in a water bath to desired temperature whereas the liver used was obtained from the refrigerator, probably at a temperature lower than the desired temperature; another reason which could have caused the occurrence of these anomalies was human error, for instance inability to use the bung to cover the conical flask as soon as possible, also because this was a group work the person taking readings from the equipments must have incorrectly read the measurements from the equipments.
Taking readings from the syringe was quite accurate even though it has an error of ±0.5cm3, but readings from the pipette was a little inaccurate because sometimes air bubble is introduced to the pipette, which must have affected the volume of hydrogen peroxide used, therefore adding to the inaccuracies of the experiment as whole.
The accuracy of all the equipments used can be calculated as in %errors. Using the difference between the measurements of the equipment I used, I can determine the inaccuracy percentage of my equipment.
Syringe
The error of the syringe is ±0.5cm3; therefore I can calculate the %error of the readings I got from the syringe.
The largest and most significant %error was 8.6% at 30oC and the smallest %error was 2.2% at 45oC (optimum temperature).
Weighing scales
The error of the weighing scale is ± 0.05cm3.This means that the reading I got from the weighing scale for the mass of the liver has an error, as I used 0.5g of liver. Therefore the %error is 0.05/0.5= 0.01
0.01*100= 1%
The error is rarely noticeable, that makes it less significant even though I still have to consider it as a %error.
Some of the things I would suggest for the improvement of the experimental procedures include (i) Liver from the same source and condition should be used throughout the whole experiment as this helps to maintain consistency (constant amount of Catalase), which in turn improves the accuracy and reliability of the experiment, if the experiment is going to take days as my experiment took then this can be done by using the same liver, after use in a day, wrap with a foil and keep refrigerated overnight, this will help reduce the amount of decomposition therefore keeping the factor constant(ii) The liver and the Catalase should be kept at the same temperature so that more reliable optimum temperature is obtained, although my experiment still showed results that supported my hypothesis, my results could have been more accurate if I had kept both the liver and Catalase at the same temperature (iii) A more accurate method should be used to collect oxygen displaced (if any) in order to reduce unnecessary lose of oxygen, and if there aren’t any one should be as fast as possible in placing the cork on top of the conical flask, as this will prevent unnecessary lose of oxygen (iv) Try as much as possible to use the same equipments, such as the syringe so that measurements will be less affected, you can make sure you use the same equipment by labeling the equipment used (v) The experiment should be repeated more and a wider range of temperatures should be used to give a more broad analysis of the experiment, so that my results are better and a more accurate average is obtained, even though I repeated the experiments three times, more repetition of the experiment is more helpful in obtaining a more reliable and accurate initial rate of reaction(vi) Try as much as possible to do a particular temperature on the same day as both the liver and the catalase will still be in the same condition for all temperatures, this will help limit the chances of getting anomalies.
Overall my experimental evidence was a reliable one, despite the effect of the errors and limitations because it supported my hypothesis and the basic knowledge about enzyme. It showed the trend of increased rate of reaction as temperatures increases up to optimum temperature, and also how rate of reaction decreases as temperatures go past optimum temperature, although the experiment will be more successful if I did this experiment again and made these changes mentioned above to it.
Finally all the improvements mentioned above are a good way of increasing the reliability of experimental evidence and also minimizing significant sources of errors, as too many uncertainties in evidence may affect conclusion drawing. The presence of this limitations and errors are quite significant because it reflected on the results table.
Bibliography
- Biology 1 endorsed by OCR
- http://www.seps.org/cvoracle/search