Substrate concentration is my independent variable, as I will be changing it’s concentration levels. The effect substrate concentration has on the rate of reaction depends on the enzyme concentration, if there is a set amount then initially enzyme activity will increase as many enzyme-substrate complexes are formed. Once however all the active sites are full converting the substrate molecules then an increase in substrate concentration will have no effect as no more enzymes molecules are being added to account for this. I will measure and observe how the substrate concentration affects the experiment by using various concentrations with a set amount of enzyme concentration.
All the above factors have an affect on the rate of enzyme activity so it’s essential to the accuracy of the results to keep all the other variables apart from the one I’m testing (substrate concentration) constant. I have outlined how I will achieve this after the discussion of each factor affecting enzyme activity.
This experiment is designed to test and evaluate the effect substrate concentration will have on enzyme activity. Specifically I aim to achieve this by having a set amount of enzyme for every experiment. Using celery, which is a rich source of catalase, will provide this enzyme. The celery will be mixed with substrate (hydrogen peroxide) and the amount of oxygen released will be recorded and will act as a measure of the rate of reaction. Firstly I will conduct a preliminary experiment to ensure I use the most appropriate method, and then I will carry out five experiments all with different levels of hydrogen peroxide.
Prediction
I predict that as the concentration of the substrate is increased the level of enzyme activity will also be increased. This change in rate of reaction will be apparent by the varying levels of oxygen produced; this level should increase accordingly with the increasing substrate concentration. This will not be the case forever though because eventually the activity on the enzyme will stay constant despite any increase in the substrate concentration. As I have explained before when the two are mixed together initially the rate of reaction steadily increases when more substrate is added because more of the active sites of the enzyme are being used which results in more reactions, so the amount of Oxygen released in a given time is higher. When an amount of substrate is added that is greater than the available free active sites the rate of oxygen produced will drop. This is caused by the enzymes already being occupied with substrate molecules, so theoretically the substrate have to ‘queue’ up to be broken into products.
As I mentioned earlier I am going to carry out a preliminary experiment to check there are no faults in my method that could alter the results. Below I have given the % of hydrogen peroxide and water that I will use in the two different concentrations.
Apparatus
Celery
Hydrogen Peroxide
Distilled water
Diagram of apparatus set-up
Ruler
Clamp
Board
Boiling Tube
Bung with delivery tube attached
Water Bowl
Measuring Cylinder
Borer
Scalpel
Tile
Safety Goggles
Stopwatch
Method
1. Measure using a ruler 1cm of celery and cut precisely on the tile using a scalpel. Using a borer (number 4) cut three circles of celery from the 1cm.Cut these three pieces in half, again using a ruler so pieces are of identical sizes, which should give you six small cylinders.
2. Appropriate solution now needs to be prepared. In total the hydrogen peroxide and water in a mixture should equal 10ml. This means that for solution 1, as it’s a high concentration of 80% substrate; 8ml of hydrogen peroxide needs to be accurately measured out using a pipette. The contents of the pipette should be squeezed out into a 10ml measuring cylinder Added to the measuring cylinder should be 2ml of distilled water also measured using a pipette. Ensure when the water and hydrogen peroxide are added together that they are mixed properly.
3. Fill the water bowl with water (doesn’t have to be distilled). To prevent spillage only fill it half full as more water will be forced into the bowl once gas starts to be given off. Fill a 100ml-measuring cylinder with water, place your hand over the top and carefully tip it upside down into the water bowl.
4. Support needs to be added to the measuring cylinder in the water bowl as if left alone it will squash the delivery tube, preventing the oxygen entering the measuring cylinder, so a clamp must be fitted on the measuring cylinder. This will also make it more level so reading results will be easier.
5. The boiling tube must also be placed in a clamp, as it cannot stand unaided; it’s easier to have the boiling tube at a slant because the bung will be easier to fit. Place the celery pieces into the boiling tube making sure that they don’t get stuck on the sides.
6. The delivery tube needs to be inside the measuring cylinder. This step is tricky, as the measuring cylinder can’t be moved above the water level because all the water would come out.
7. Make a note of the initial starting point of the water in the measuring cylinder. The solution of hydrogen peroxide now needs to be added into the boiling tube, but you must be prepared to start the stopwatch and place the bung on top of the boiling tube immediately after all the solution has been added o the celery, so you may need help at this stage.
8. Every minute the water level in the measuring cylinder needs to be recorded, you should do this for ten minutes.
9. After you have completed the experiment, all equipment must be washed and put away.
Results from preliminary experiment
Method One Amount of oxygen evolved (cm)
This method involves measuring the amount of gas given off by observing how much water is displaced in a measuring cylinder. While the preliminary results were near what I would expect, it does have it’s shortcomings, the main disadvantage being the fact that once the hydrogen peroxide solution has been added to the celery in the boiling tube, the bung needs to be attached. Obviously some time will elapse before this happens and this will allow any oxygen being produced to escape. There is however another method I can try that would be more accurate as oxygen wouldn’t be able to escape, and this is by using a suba seal, which would make the set-up airtight, below I have outlined of how to carry it out, it’s shorter than the first method as some of the steps are simply repetitious of method one.
Apparatus Diagram of apparatus set-up
Celery
Hydrogen peroxide
Distilled water
Pipette
Tile
Scalpel
Borer
Gas syringe
Suba Seal
Needle
Syringe
Stopwatch
Conical Flask (adapted for delivery tube)
Method two
1. Prepare celery as directed in method one.
2. Prepare hydrogen peroxide solutions as outlined in method one.
3. Use the syringe by placing the end in the hydrogen peroxide solution and pulling the extending arm so that the air pressure ‘sucks’ the solution into the syringe.
4. Place the celery into the conical flask, and then put the suba seal into the top of the conical flask making sure that is as far down as possible so that no gas can escape. 5. The top of the suba seal now needs to be folded down, this can be quite tight as the suba seal is rubber, and so getting someone else to hold the conical flask may be helpful.
6. The gas syringe has to be held in place by a clamp. The positioning of this is also important for it must be level otherwise gravity will have an effect on the results.
7. The conical flask and gas syringes need to be connected by a delivery tube; using fairy liquid here may be useful as it can be a bit tight. You need to check the set-up is airtight by gently pulling the gas syringe, if it is brought back to it’s original place it means it’s airtight, if not the apparatus needs to be double checked.
8. Put a needle onto the end of the syringe, and push this into the suba seal, as quickly as possible you need to squirt the liquid into the conical flask. Once this has been done record the level of the gas syringe, and do so every minute until ten minutes is up.
9. Wash and tidy up all the equipment you used.
Safety
Hydrogen peroxide will be used throughout this experiment, so goggles must be worn constantly to avoid any damage because hydrogen peroxide is a corrosive chemical. In addition to this gloves must also be worn to protect skin, as minor spillages are common. It is essential to wear a laboratory coat to prevent any chemicals damaging clothing and reaching the skin underneath. In both of the methods a scalpel is used to cut the celery, and if these aren’t used sensibly they could cut the skin. Most dangerous however is the needle in method two as the syringe contains hydrogen peroxide. Because of this fact the needle should only be put on the syringe just before it’s needed and taken off immediately afterwards.
Method Two Preliminary Results
Amount of oxygen evolved (cm)
Before I conducted these two different methods I assumed that method two where the gas syringe was used would produce more accurate results, because it provided an airtight atmosphere so no gas could escape. Whereas during method one, some time would elapse between adding the hydrogen peroxide solution to the celery and placing the bung on top of the boiling tube and this would allow any oxygen produced to escape. However it would seem that I achieved the most accurate results from method one. Obviously there was a problem with experiment two because oxygen should be steadily produced for more than ten minutes. I made several attempts using method two but despite my best efforts I could not get the gas syringe to move after a couple of minutes. I tried several ways to improve it including using different gas syringes, changing the amount of hydrogen peroxide solution and using fairy liquid to help move the delivery tube as far up as possible. I can only conclude from these results that for the real experiment I will have to use method one as it produced results that would be expected i.e. higher concentration gave more oxygen than lower concentration. Although perhaps if method two had of been suitable to use, it may have produced more accurate results, I can improve method one by using an inverted burette instead of a measuring cylinder as the markings are easier to read which would give me more precise readings of the results.
I now need to consider what would be an appropriate range of substrate concentration. The range needs to be varied enough so that I can analyse the results as thoroughly as possible. I will be using five different concentrations, that will be approximately very high, very dilute and three in between at sensible values. The table on the next page what these will be.
Hopefully these percentages will give me varied results that will enable me to either support my prediction or state why it was not correct. Not only will I carry out these five experiments but also I will have to use repeats. Repeats are a way of making sure your results are reliable for if they match each other then you know they must be somewhat accurate. I think completing three repeats for each different concentration will allow me to identify any anomalous results.
As I will be using 10ml of each different concentration, the percentage concentrations need to be divided by 10 to work out the amount in ml. For instance take solution 3:
60 = 6 ml of hydrogen peroxide
10
Results from 100% hydrogen peroxide solution
Amount of oxygen evolved (cm )
Results from 80% hydrogen peroxide solution
Amount of oxygen evolved (cm )
Results from 60% hydrogen peroxide solution
Amount of oxygen evolved (cm )
Results from 40% hydrogen peroxide solution
Amount of oxygen evolved (cm )
Results from 20% hydrogen peroxide solution
Amount of oxygen evolved (cm )
Analysis of results
While I have set out all the results I achieved above, I think it might be easier to analyse them in a table that relates the concentration to the total amount of gas evolved, which I have done below.
On the whole these results are what I would expect, and certainly follow mainly what I predicted. Apart from the results for 80%; as the concentration of hydrogen peroxide increased so to did the rate of reaction. The rate of reaction for this experiment is determined by how much oxygen is produced by the reaction.
The first major step in terms of analysing the results and determining whether my prediction is supported by my results is to draw an appropriate graph (this is shown on the next page).
To enable comparisons to be drawn between the results of different concentration levels, I have drawn all the lines on the same graph. These lines are in different colours to allow for easy recognition. The orange line showing the results for 80% clearly has the steepest gradient, meaning it reacted the fastest, this is perhaps to be unexpected as more substrate would surely mean the active sites in the enzyme (celery) would be continually busy converting it into products. If the 100% and 80% had produced similar results the explanation would simply be that the increased concentration had no effect because the enzymes were already working at V-max and excess substrate would theoretically have to ’queue up’ until an active site was free. However because the 80% continually produced more oxygen than 100%(shown through repeats) I’ll have to explore other explanations (in my evaluation) as this particular results doesn’t support my prediction.
Excluding the 80% results, the rest of the graph reasonably shows what I would expect, the 100% contains the most substrate molecules and so it’s only logical to presume it would have the highest rate of reaction as it’s more likely to bind with an enzymes active site than say a solution containing 20% hydrogen peroxide as there are less substrate molecules. Purple and red lines represent the 60% and 40%. It’s difficult to distinguish between the two as they have such similar, lines of best fit. By looking at the results tables though, it’s possible to see 60% produced marginally more oxygen, so this is in keeping with what was predicted. The reasons why these two sets of results are so close is related to the above discussion over the 80% being faster than 100% and will be explored in my evaluation. The 20% gives the lowest results, as the majority of the solution is water, and water doesn’t cause the reaction to produce oxygen so invariably the amount of oxygen produced should be low.
The beginning of the reaction is the only time we can be sure that any differences in the amount of oxygen produced are only caused by the fact they all have varying levels of hydrogen peroxide. Once the reaction has been going for awhile, the substrate amounts begin to vary, as products are being converted at different rates, this means the most accurate way of comparison is by looking at the initial rate of reaction. To measure the initial rate of reaction I can simply read of my original graph the amount of oxygen produced after 30 seconds, and then plot these on a graph against the enzyme concentrations. This graph is shown below.
The graph on the page before is interesting in that it, suggests rather than the 80% being faster than average (as I presumed) it’s more likely from analysis of the graph that the 100% was slower than average. Although this could still be for the same reasons I listed earlier e.g. the celery used for the hydrogen peroxide in the 100% experiment was older than that used for the 80% experiments which affected the rate of reaction e.t.c. The 40% and 80% are actually on the line, while the 20% and 60% are very near and wouldn’t be counted as anomalies. This graph shows the initial rate of reaction increases linearly.
I’m now going to present the raw data in a pie chart. There are 360 degrees in a circle so to find out how many degrees each concentration has, I need to add the total amount of oxygen evolved from each different concentration together:
19.0 + 20.9 + 21.2 +27.7 + 23.9 = 112.6
And then divide this number by 360, to work out the ration between cm and degrees.
360 = 3.2 degrees
112.6
Now the amount of oxygen evolved needs to be timed by 3.2 to determine how many degrees each concentration should have out of the pie chart.
20% - 3.2 x 19 = 60.8
40% - 3.2 x 20.9 = 66.9
60% - 3.2 x 21.2 = 67.8
80% - 3.2 x 26.0 = 83.2
100% - 3.2 x 23.9 = 76.5
Pie chart to show the amount of oxygen produced from varying concentrations of hydrogen peroxide
I have drawn error bar graphs for all the different concentrations. They couldn’t all fit on one graph however because the bars would overlap, so I’ve drawn two graphs showing alternate lines. Due to the limited space on the graph paper, the values on the y-axis are slightly squashed, and this makes it difficult to determine any major differences in the error bar graphs for different concentrations as they all seem relatively similar. This would suggest that they are fairly accurate, as the same amount of error was there throughout all the different concentrations. If any the 60% seems to have the largest error bars especially nearer the end, and if you look at the results for 60% you will see why, for one repeat is much faster than the other two. As this repeat was the first one conducted I can only conclude that the experiments were carried out on two different days, which would explain why the latter two show similar results.
Evaluation
I feel I have carried out this experiment to the best of my ability, and therefore have produced accurate results. While the majority of these support my prediction, there are some sets that don’t; this mainly concerns the 80% solution of hydrogen peroxide where it seems it was faster than expected or alternately it could be said the 100% solution was slower than previously anticipated. Either way there are several explanations for this but they all come down to the fact that it wasn’t possible to conduct all these experiments at the same time.
These results were collected over a number of different biology lessons and the day and time of these vary, so inevitably the temperature couldn’t remain constant throughout. Although I did mention earlier that this could prove a problem there’s little else I could do apart from measuring it using a thermometer. It’s far more likely though that the anomalies were connected to something other than temperature, for instance with a class of 25 students, hydrogen peroxide ran out rapidly so new bottles were constantly being used, and slight differences between batches may have affected the results, not only this but the age of the solution may have been a contributing factor as generally left-over hydrogen peroxide was kept until the next lesson.
As with the hydrogen peroxide several packets of celery were used throughout, and again the age of the celery varied, and this may have had an effect on the enzyme’s within. In the conclusion I think it’s probable the large differences experienced between the total amount of oxygen produced at 80% and 100% was due to the variability of the equipment. The only way I can suggest to eliminate this limitation would be to perform all the experiments for the different concentration levels at the same time, as this would ensure the hydrogen peroxide and celery were all the same age and from the same batch, although this would require a lot of assistance to carry out and could prove very hectic.
As I was using an inverted burette I think I can be quite confident of the accuracy of my measurements, for burettes are very precise and have clearly marked values along the side. Apart from reasons already discussed, the most significant limitation of the method I used is the lack of an airtight environment. Using a gas syringe connected to a suba seal is the obvious solution to this problem, but as I experienced problems with this during my preliminary experiments I thought it wasn’t practical to use, but many others from my class were able to produce reliable results from it, so given the chance where I could be assured the results were reliable I would use a gas syringe.
Improvement on accuracy of results could be achieved by minor changes such as increasing the number of repeats used, so that the average was worked out using more sets of results and overall improving it’s reliability. As oxygen was still being produced at a steady rate when I terminated the experiment after 10 minutes, it could prove useful to continue recording the results for a further 10 minute as this might mean more trends could be identified between the different concentrations levels and the analysis could be covered in more depth.
I have focused mainly upon the discrepancy between the 80% and 100% concentration levels but there are also other points for discussion. If you look at the table displaying the results recorded for the 60% solution of hydrogen peroxide solution you’ll see that the first repeat produced reasonably more oxygen than the second two repeats, I think this can be explained using reasons I outlined earlier, the first repeat was carried out on a different day to the other two so the hydrogen peroxide and celery would have been different and this lead to a discrepancy. I believe that the first result is probably most accurate. This also explains why the lines on the graph for the 60% and 40% are so close as the 60% should probably have been higher.
