Factors that affect the of an enzyme
There are 3 main factors that affect the rate of reactions of enzymes:
- Temperature
- pH
- Concentration of enzyme/substrate
Temperature:
At low temperatures, the enzymes and substrates have low kinetic energy so they are moving around slowly and only a few reactions occur. When the temperature is increased, the Kinetic Energy also increases; therefore, the enzymes and substrates have more energy so they move around faster. More movement leads to more collisions between enzymes and substrates; thus, more enzyme-substrate complexes can be formed. The rate of reaction continues to increase up to a certain temperature. This is called the optimum temperature and the rate of reaction is at its peak. Most enzymes have an optimum temperature of around 37oc. But when the temperature is too high, the weak bonds that hold the enzyme in its tertiary structure can be broken so this causes the enzyme to denature. The graph below shows this:
pH:
pH is a measure of the concentration of hydrogen ions (H+), or the number of Hydrogen ions in a certain volume. The concentration of hydrogen ions affects hydrogen bonds and ionic bonds in the tertiary structure of the enzyme. A change in these bonds will result in a change in the specific 3D shape of the enzyme. When the shape changes, the enzyme denatures. The optimum pH for most reactions is pH7 (neutral). The following graph shows the effect of changes in pH in a reaction:
Concentration:
The concentration of the enzyme and substrate affects the rate of reaction. The higher the concentration of the enzyme/substrate, the more enzyme/substrate there is, therefore there’s more chance of the enzymes and substrates colliding and forming enzyme-substrate complexes.
Gas produced:
In the reaction between hydrogen peroxide and Catalase, a gas is produced. My idea was that the gas could be one of the following: Carbon Dioxide, Hydrogen or Oxygen. Because they are the most common gases produced in reactions. To identify this gas I used 3 basic tests.
Oxygen-if the gas is oxygen it will relight a burning splint that has been put out. My results were negative so oxygen was ruled out.
Hydrogen-to indicate whether or not the gas was hydrogen, I used ‘the pop test’. There will be a ‘pop’ sound when a burning splint is placed near hydrogen gas. This result was negative so by the process of elimination, I declared that the gas produced in the reaction is Carbon Dioxide (CO2).
PRELIMINARY WORK
In order to decide what methods I would use to record the rate of reaction between Catalase and H2O2, I conducted a series of preliminary experiments. From my basic scientific knowledge, I realised that in an experiment involving enzymes, heat energy is produced and released in the form of gas (exothermic reaction), so I decided that a good method of measuring the rate of reaction was to measure the amount of gas produced on reaction. I varied the amounts of enzyme and substrate used in order to decide what values will suit my real experiment the best.
I will not go through the method and apparatus etc of the preliminary work because it is near identical to the methods used in the real experiment and I will highlight those later.
These are my results for the preliminary experiments that I carried out:
Analysis of preliminary results:
After I did the 1st 3 experiments, I realised that an excess of 100ml of gas is produced with ease so I recorded the time taken to reach 100ml, and this time would give me an idea of the rate of reaction. I decided to use 20cm2 of solution rather than 10cm2 because it will allow me to use a larger variety of concentrations. I decided to use 1g of yeast rather than 0.5g because 0.5g took quite long to produce 100ml of gas even at 100% concentration so I thought that if I use more enzymes, the rate of reaction will increase. And I did not use more than 1g of yeast because the experiment would be too quick and this would make it difficult for me to record accurate results. Preliminary experiment 5 (PE5) was done to check if all concentrations produced over 100ml of gas and I found from this experiment that this is not the case.
As I am investigating the effect of concentration changes on the rate of reaction, I will have to keep the PH and the temperature constant throughout the experiment and will have to vary the concentration of the solution.
I will keep the temperature constant by conducting all my experiments under standard room temperature (21oc approximately) because if my temperatures varied, this would have an effect on the results because enzymes work differently at different temperatures.
I will keep the pH constant by keeping the yeast in a buffer solution if possible.
PREDICTION
I predict that as the concentration of H2O2 increases, the rate of reaction will also increase. My reasoning for this is because when the concentration of the substrate (H2O2) is high, there is more collisions with the enzyme molecules therefore more enzyme-substrate complexes are made. When more reactions happen, more gas will be released. When the concentration of the substrate is low, there is a high proportion of enzyme compared to substrate and not all enzymes active sites will be occupied due to the lack of substrate molecules available. This will make the rate of reaction decrease.
HEALTH AND SAFETY
There are some important health and safety issues that I will need to consider and take out. These are:
- Wear Goggles to ensure safety of the eyes because hydrogen peroxide is corrosive and irritant and high precautions must be taken to ensure that it does not get into contact with the eyes.
- Wear a lab coat to prevent chemicals from affecting clothing.
- Put all bags and coats etc on the side to ensure that I will have a clean and clear working space.
APPARATUS
This is the list of apparatus that I will need for my experiment:
- Metal Clamp Stand and Clamp
- Gas Syringe with bung and cork
- Conical Flask
- Measuring cylinder
- Stop-watch
- Test tubes
- Test tube rack
- Beakers
- Weighing scale
- Substrate-Hydrogen Peroxide Solution
- Enzyme-Catalase stored in Yeast
- Water
- Sticky labels
- Thermometer
METHOD
- Wear plastic goggles and a lab coat for safety reasons
- Set up the apparatus as shown below. Make sure to thoroughly wash and/or clean the equipment before use to avoid contamination in the experiment.
- Measure out the different concentrations of hydrogen peroxide solution using the following table as a guide. To measure out the concentrations, pour the required amount of solution from its designated beaker into the measuring cylinder until the required amount is reached. Place the solutions in separate test tubes and label them using an appropriate method. In my case I used sticky labels.
- Record the room temperature and measure out 1g of yeast with the weighing scale. Be sure to measure it out accurately to prevent defects in the results.
- Take the test tube with 100% concentration and pour the contents into the conical flask.
- Add the yeast to the conical flask and immediately seal the conical flask with the cork and start the stopwatch. To make sure that all the yeast is reacting with the substrate solution, swirl the conical flask slowly so that all the yeast gets in contact with the solution. Note that too much swirling will increase the kinetic energy of the molecules so the rate of reaction will increase, therefore take caution in ensuring that the conical flask is not swirled excessively.
- Pay attention to the gas syringe and stop the stopwatch as soon as 100ml of gas is produced and record the time taken in a table. If 100ml of gas is not produced then record the final amount of gas produced. The reason that I’m measuring up to 100ml of gas is because the gas syringes that are available to me have a maximum of 100ml on the scales.
- Wash and dry out the conical flask and reset the stopwatch. Then repeat steps 5-7 but change the concentration each time. The 0% concentration of hydrogen peroxide solution is designed as a control solution to show that at 0% concentration no reaction occurs
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As a means of getting the most accurate results possible, repeat steps 1-8 twice to get a 2nd and 3rd set of results. Then make an average out of these results using the following method:
E.g. if the 1st result is 10 seconds, the 2nd result is 15 seconds, and the 3rd result is 12 seconds, add all 3 results together and subsequently divide the resulting answer by 3 to gain an average:
10+15+12=37.
37/3=12.33 so the average is 12.33 seconds.
- Record all results in a table and plot graphs to represent the data.
RESULTS
Before each of my experiments, I recorded the temperature of the room with a thermometer and it was 210C in all cases.
After I obtained my results, I realised that the results for 20% and 30% concentrations did not produce my target of 100ml of gas, so I decided to exclude these results from my final analysis.
ANALYSIS/CONCLUSION
My results clearly indicate that as the concentration of substrate increases, the time taken to produce 100ml of gas also increases. The amount of gas produced indicates the rate of reaction.
In my experiment, my prediction was proved to be correct. I predicted that as the concentration increases, the rate of reaction will also increase and this hypothesis is deemed correct through my investigations.
Graph 1:
This graph is very simple. It simply shows how much gas is produced at each concentration in each of my 3 attempts at the experiment. It is clear that all concentrations from 30%-100% produce 100ml of gas. Any concentration below that has been ignored in my experiment because the reaction is not sufficient enough to compare with the rest of the concentrations.
Graph 2:
In general, there is a clear trend in the graph. As the concentration increases, the time taken to produce 100ml of gas decreases. This indicates an increase in rate of reaction as the concentration increases. This trend is apparent for all the concentrations apart from the first result at 30% concentration. Between 30% and 40%, the time taken to produce 100ml of gas increases rather than decreasing like the other concentrations. Due to this abnormality in my results, I will consider the result for 30% concentration as an anomalous result. This anomalism is indicated on the graph.
The reason for the general increase in rate of reaction is that as the concentration increases, there are more substrate molecules available so this acts as more ‘food’ for the enzymes active sites. The active sites are filled up much more quickly when the concentration is high.
Graph 3:
This graph shows the trend for the results I obtained when I repeated the experiment for the first time. The trend observed in graph 1 is followed, and the reasoning behind this trend is the same. The main difference between this set of results and the previous set of results is that the range of results (highest time taken-lowest time taken) is much wider. The reasoning behind this is that the second set of results was taken at a different time to the first set of results. In this experiment I used different apparatus to the first attempt and due to limitations (e.g. shortage of equipment) the results were different. I noticed that the bung I used was very long compared to the bung in the first set of results. This makes a longer distance for the gas to travel before it gets into the gas syringe. But most importantly, this slight defect does not have a major effect on my results because the general trend is the same and this is the most important aspect.
Graph 4:
This is the graph for my second and final repeat of my experiment. Again, the same trends were observed but the major difference was that all my times taken were very small compared to my first 2 experiments. The reasoning for this is that after my first 2 attempts, the yeast that I was using was fully used up so a new, unused tub had to be put in use. This new batch of yeast made my results take place very quickly thus giving me the impression that the first tub of yeast that I used was maybe tampered with in some way. But again, this imperfection was ignored because the general trend of rate of reaction increasing as concentration increases is continued. One of the points on the graph (circled in red) is not included because without it, I get a better curve on the graph. The curve is designed to be the line of best fit and including the anomalous point gave my curve a slight deformation so I decided not to include it.
Graph 5:
This is the most important graph because it shows my final, averaged results. From this graph, it is clear to see that as the concentration of the substrate increases, the time taken to produce 100ml of gas decreases. This trend is explained earlier in words but I will explain it diagrammatically to make it easier to understand.
High concentration of substrate:
= Enzyme
= Substrate
There are lots of substrate molecules so the active sites are all occupied and producing reactions. And when a product is formed, the enzymes will not be used up so the remaining substrate molecules can be used up until there is none left.
Low concentration of substrate:
There is a lack of substrate so most active sites are empty and fewer reactions occur.
ANOMALOUS RESULTS
The time taken to produce 100ml of gas at 30% concentration in my first set of results was an anomalous result so I ignored it.
The result for 50% concentration in my 3rd attempt was also ignored on my graph of results because if it was included, the curve of my graph would be slightly deformed. As all the curves of my graphs are designed to indicate the best fit of the points on the graph, I decided not to include this result because without it I got a much smoother curve with a much clearer trend.
EVALUATION
Limitations:
- There is a slight delay when I put the yeast into the conical flask full of substrate solution, then place the cork on the conical flask and finally starting the stopwatch. I only used a stopwatch, which relied on my reaction to stop the watch. Everyone’s reaction times are different so it is not particularly the most accurate way of timing but it was the best method available to me. This will slightly affect all the results for each individual experiment but as I carried out all the steps in the same way, it should not make any negotiable difference to the overall result. The problem of the delay between pouring in the Yeast, bunging the conical flask and starting the stopwatch could have been limited by getting another person to start the stopwatch when the yeast was added to the substrate.
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It is also impossible to precisely measure out the amounts of Hydrogen Peroxide and Water each time. As the scale on the measuring cylinder shows the measurement to the nearest 1cm3, the solutions that I used should be correct to the nearest cm3. This is only a slight imperfection and will not have much of an effect on my results. It would also be difficult to gain even more accurate measurements due to limitations in apparatus.
- I only had limited time to complete my experiment. I repeated my experiment another 2 extra times but I feel that the experiment could be repeated more times to help get rid of any anomalies. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments.
- The length of the bung in each of my experiments differed. This means that there is a different length for the gas produced in the conical flask to travel in each of the experiments. This affected my results because the times that I obtained in each of my 3 attempts were very varied in terms of the range of results. But overall, the same basic trend was observed in all my experiments so this defect is not major. But if I were to repeat the experiment, I would ensure that I used the same bung each time.
- The yeast that I used in each of the experiments was different. The reason for this is because I carried out each of my experiments at different times and the tubs of yeast made available to me could have changed each time. For my final repeat, I used a new tub of yeast and this gave me much faster rates of reaction than the first 2 attempts. My assumption is that the first batch of yeast must have been tampered in some manner by any of the previous users, because it had a powdery appearance whereas the new tub that I used was in granules. This imperfection did affect my results but most importantly did not affect the general trend of the results. If I were to repeat my experiment, I would use the same sample of yeast for all my experiments.
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It is very difficult to control the amount of swirling of the conical flask while the reaction is taking place. Swirling excessively will result in an increase of kinetic energy of the enzyme and substrate molecules therefore they will move around faster and collide more, so this will cause the rate of reaction to increase. No swirling could lead to some of the yeast not getting into contact with the substrate solution and this would cause the reaction to be incomplete. The method I used to overcome any abnormalities and to get a fair test was to swirl the conical flask 3 times for 360o each time and maintaining a constant speed of swirling. Obviously the amount of swirling cannot be exactly the same each time but I did my best to swirl the conical flask by the same amount each time.
ALTERATIONS
I only made a slight alteration to my method. In my method I mentioned that I will control the pH by storing it in a buffer solution, but I realised that there was no need for this as the pH would remain constant anyway.