Independent Variable
As the investigation is based upon the effect of the change in the concentration of activator therefore it should be kept as the independent variable. The reason being, that the effect of the activator concentration can only be measured when the Activator concentration is being varied. Therefore I will be using the following concentrations for my experiment. 0.5%, 1%, 1.5%, 2%, 2.5% and 3%
Fair Test
Certain measures have to be carried out in order to insure that a fair test is carried out. Which are as follows: -
- The experiment for each concentration will be carried out three times so that to insure the results are reliable and to minimise the chances of error in the recording of the results.
- All the solutions use din the experiment will be starred before use in order to insure that the solvent is evenly spread through out the solution, keeping the concentration of the solution the same.
- A separate syringe is used for each of the solutions to minimise the chances of contamination.
- While taking all measurements the readings should be taken at right angle to the scale of the apparatus, reducing the chances of inaccurate measurement of the volumes.
- Also insuring that all apparatus use din the experiment is completely dry as a slight variation the concentration of the reactant will affect the rate of reaction adversely therefore giving us anomalies.
Prediction
I predict that varying the concentration of the activator will affect the rate of reaction by affecting the activity of the enzyme.
As in my investigation I will be increasing the concentration of the activator therefore the rate of reaction will increase. Thus I can say that the activator concentration and the rate of reaction will be directly proportional.
The reason being that the activators work by binding to a part of the enzyme.
They bind at a little pocket in the structure of the enzyme, which exists {away from the active site} where the activator binds. Upon binding with the pocket it comes into contact with the atoms present. Therefore binding with the structure causing it to alter the shape of the Enzyme and provide stability. It not only provides stability but also strengthens the structure of the enzyme by doing so it increases the activity of the enzyme. As the bonds forming the enzyme are strengthened by the addition of an inorganic ion, this makes the active site harder to alter therefore allowing the substrate to attach to the active site more readily and increasing the chances of collision and then breakdown. This is achieved by the inorganic ion attaching to its site and therefore altering the charge of the enzyme; by doing so it speeds up the reaction therefore increasing the rate of breakdown.
Preliminary Experiment
Before starting the actual experiment it is very important to conduct some preliminary work in order to determine weather or not the various volumes and concentrations of substances which are to be used in the main experiment are suitable or NOT.
Therefore two simple investigation swill be conducted as follows
- Determining suitable Buffer solution
For this experiment only the buffer solution will be varied and all other factors will be kept constant in order to insure a fair test. Therefore for this experiment I will be using the following the following buffer solutions pH 2, pH 3, pH 4, pH 12. I will be following the same method as stated for the main experiment but the only difference will be that the Activator will be not be used and the ph will be varied. The results are shown on the result table in Implementation.
- Determine Suitable Volumes of Enzyme and Substrate
From the results obtained in the first pilot I will use the Buffer solution and conduct the experiment without using the Activator. I will be using the following volumes of Fungal Amylase 2cm3, 3cm3, 4cm3 respectfully and the following volumes of starch 1cm3, 2cm3 and 3cm3 respectfully. The results for the experiment are attached with the actual results.
Apparatus
- 4 (5ml) syringes
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4 50cm3 Beakers
- Test tube holder
- Stop clock
- Test Tube Rack
- China Graph pencil
- 3 Stirrers
- 2 Electrical Heaters
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2 100 cm2 Beakers
- pH Meter at pH 4.5
- Distilled Water
Method
- The apparatus for the experiment will be set as shown above.
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2cm3 of Buffer solution was added to a test tube
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Followed by 2 cm3 of 0.5% calcium ions (activator)
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3cm3 of 2% starch and 4 cm3 of 1% fungal amylase was added to the same test tube
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This mixture was then placed in a water bath at 50 oC for 6 min
- Then Benedict’s reagent was added to the test tube
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It was then boiled at 100 oC
- And the time taken for the Benedict’s reagent to turn the solution red was measured.
- The experiment was repeated for each concentration 3 times.
Safety
- All chemicals should be handled with gloves and goggles on
- The test-tubes should be held using a test- tube holder when heated (not by hand)
- Any spillage must be reported to the Teacher or Lab Assistant immediately
- Safety labels on Chemicals must be read and the necessary precautions must be taken for all chemicals.
- All wet apparatus must be put on tissues to dry rather than directly on the table.
- Stoles must be under the table at all times and bags and coats should be at the front.
- Lab coat must be worn at all times
- When metal plate is on it should never be touched by hand.
Analysis
The Practical aspect of the investigation provided me with a raw data table from which I obtained the averages of the time taken for the breakdown of the substrate {starch} by the enzyme {fungal amylase}. From the averages, which I obtained, I finally derived the rate of reaction {1/Time}.
By having the rate and the known concentrations for the enzyme activator {Ca ions from CaCl2}, I was able to plot a graph of rate against enzyme activator concentration.
From the graph plotted, {refer to previous page} the first piece of data is crucial as this shows the rate of reaction of the enzyme without the presence of the activator ions. Therefore, according to my stated hypothesis any point that follows the initial point should be greater in value as the activator should have the effect of increasing the rate of reaction. This expected trend can be clearly seen on the graph, that as the percentage concentration of the enzyme activator increases so does that rate of reaction. By looking at the best fit line we can see that the rate of reaction is increasing by approximately 0.1 with an increase in 0.5% of activator concentration. The reason for using the best fit line rather than the exact averages is due to the anomalies at activator concentration of 1 % and 2%. These anomalies would affect the rate of increase significantly as they do not lie on the line of best fit. The reason for these anomalies being that I was rinsing the syringe every time I used it to measure volumes, there may have been some water left behind which affected the concentration of the activator, As I was stirring all solutions before use, there may be some miscalculation in stirring as there is no definite way to determine if a solution is being stirred precisely the same as the others. Thus it is important to use the best fit line to interoperate the results and derive conclusions rather than the actual results. Therefore, I believe it would be fair to say that enzyme activator concentration is directly proportional to the rate of reaction.
The reasoning behind this trend strongly relates to the theory of enzyme activators. The theory stating that activators in a way act as stabilisers {via the binding of the Ca ions to the enzyme makes its structure more rigid and less likely to denature} hence making them more thermo-stable, able to work at higher temperatures. As the enzyme, being examined is fungal amylase {denaturing begins close to 60°C} the experiment itself was carried at 60˚C therefore the low rate of reaction at 0% activator concentration as the enzyme is being denatured, as the activator is added the rate of reaction increase significantly from 0.178 at 0% concentration to 0.483 at 1.5% concentration, similarly the rate also increases with approximately the same ratio from 0.483 at 1.5% to 0.800 at 3%, By comparing the two set of readings taken from the graph we can clearly see a difference of approximately 0.3 on the rate with the increase in the concentration by 1.5%, by keeping all other variables constant and having such a increase in the rate of reaction proves that the activator concentration produces a greater volume of broken down substrate therefore showing that the rate of reaction has Increased. This increase in rate of reaction is due to the increasing number of atoms of the activator colliding with the binding site as the concentration increases.
Therefore I can say that I have achieved the desired results from the investigation, as my prediction has been proved right.
Evaluation
After conducting the investigation and then analysing the results, I found that the experiment as a whole was quite successful. I can say that because the Graph produced after interpreted the results showed the desired results for the investigation. However there were two anomalies in the results obtained which did not effect the results of the experiment as any conclusions made were taken by interpeting the line of best fit. The two anomalies obtained were at 1% concentration and at 2%. There are a number of factors that could have lead to these anomalies.
- The first and for most being that through out the experiment I was using the time taken for the colour change of the solution due to the breakdown of starch in Benedict’s reagent.
- Secondly As I was Using three test-tubes through out the whole investigation therefore I had to wash the test-tubes after every use this allowed some water to be left in the test-tubes even after leaving them to dry for a short while this resulted in the concentration being varied therefore NOT giving the expected result
- Before using the solutions it is important to stir the solutions in order to allow equal mixing of the solute and the solvent. During the experiment I may not have stirred the solutions properly before use therefore I am getting the anomalies in my results.
Further more the method of the experiment can be improved by using smaller volumes of the reactant as with these volumes the increase in the rate of reaction is quite significant but would be more suitable if smaller volumes of the reactant were used and greater volume of the Activator would be used (not in such quantities that the rate of reaction is very difficult to measure). Another means of improving the experiment would be to use a greater range of concentrations as this will allow more reliable results to be taken and the line of best fit will be more precise and accurate. The accuracy of the results can be increased by increasing the number of trials therefore enabling a more precise average to be taken, further reducing the chances of an anomaly.
The investigation could be carried out by using chloride ions instead of calcium ions, this will enable us to conduct the experiment at lower temperatures and also allow us to find the effect of the activator more precisely as it will be easier to conduct the investigation because the Buffer solution required is pH7.
Over all my investigation was fair and the desired results were produces, my prediction was right therefore showing an increase in the rate of reaction.
Bibliography
Title Author/s
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Essential AS Biology Glenn and Susan Toole
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Biological Science 1&2 2nd Edition N.P.O.Green, G.W.Stout, D.J.Taylor
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Biology 3rd Edition Arms& Camp
CDs
Compton Interactive Encyclopaedia 1998 Edition
Internet
