This is a simple way of describing how specific an enzyme is for its substrate. Each enzyme is a protein which is a polypeptide chain folded into a complex 3 dimensional structure. Part of that structure contains the active site where the enzyme binds to the substrate to form a chemical reaction. This theory was suggested in 1984 by Emil Fischer.
The graph then levels off as the rate of reaction becomes constant. Reason being, in a plant cell a limited number of water can enter the cell because the protoplast shrinks, but the cell wall restricts the cell from bursting. The vacuole is full of water, as water can no longer enter the cell. The cell becomes turgid as the cell is fully inflated with water. (Advanced Biology)
Catalase is one of the fastest enzymes with a turnover number in the millions. This shows the speed of the decomposition of hydrogen peroxide. This is the number of substrates converted into products by an enzyme molecule per minute. (Biology-a functional approach)
This enzyme is also capable of detoxifying hydrogen peroxide and prevents the formation of carbon dioxide.
Catalase is synthesized within the cell from amino acids. It has four sub-units; each sub-unit carries a heme group. In the middle of the heme group sits an iron atom. This then breaks the bonds of the two molecules of hydrogen peroxide to release two molecules of water and one molecule of oxygen gas. Here is what a heme group looks like:
Each enzyme is specific for a particular reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The active site interacts with the substrate so that the any substance that blocks or changes the shape of the active site affects the activity of the enzyme. (www.madsciorg)
Variables That Are Going to be Kept Constant
There were a number of variables in this experiment which can alter the rate of reaction of the enzymes. Due to this they need to be kept constant. They are as follows:
pH
To control the pH value I will use a pH buffer. This will be kept constant throughout the experiment as I will use 3 drops of the pH buffer.
As the pH increases the enzyme will lose its shape as it has lost its positive H ions. If the pH is decreased the enzyme will gain positive H ions. The enzyme would not be able to fit into the active site as it would be denatured, and as a result would be dysfunctional.
Substrate Concentration
The substrate is hydrogen peroxide which I’ll keep constant at 10cm³.
As the substrate concentration increases so does the rate of reaction, until there are no more active sites for the substrate to occupy as all of them are full, resulting in the reaction having no increase. Here is a graph to support this particular point:
Enzyme Concentration
I will keep the mass of the kidney beans constant. I will use 5g of kidney beans.
As the enzyme concentration increases so does the rate of reaction, until the substrate molecules are limited. The active site of the enzyme can be occupied again and again. The rate of reaction will be proportional to the enzyme concentration. (See graph below)
Temperature
More heat over 40ºC+ breaks the hydrogen, ionic, and hydrophilic bonds. These tertiary structures hold the enzyme structure together.
I will keep the water bath at 25ºC by using a Bunsen burner. The temperature will therefore be controlled.
Generally as the temperature increases the chemical reactions speed up. At low temperatures the reaction is slow as there are not enough collisions, due to the molecules moving slowly. At high temperatures there are more collisions, so the molecules are going to be travelling faster. There will be an increase in kinetic energy. There fore, the substrate molecules will enter the active site more often. This is called ‘The Collision Theory.’
But an enzyme molecule can begin to lose shape as the hydrogen bonds break. This enzyme is said to be denatured. The temperature at which an enzyme catalyses a reaction at the maximum rate is called the optimum temperature.
The temperature of the hydrogen peroxide will be kept constant at 25ºC. By doing this equilibrium can be reached.
Time for Kidney Beans to be Grinded
The time that I grind the beans for will be 10 seconds. This will be kept constant throughout the experiment. The reason for this is because so there does not be indifference in the surface areas of the kidney beans, keeping in mind about the fair testing.
Timing for Each Experiment
The time taken to measure the volume of oxygen will be kept constant at 2 minutes. This is another matter to keep the experiment fair, so I can get accurate results.
Variables That Are Not Going to be Kept Constant
Time for Kidney Beans to be Soaked
I will be using a variation of soaking times ranging from 0 hours to 5 hours. Therefore the length of soaking will not be kept constant.
Apparatus
The apparatus that I will use in this experiment are:
- 20cm³ Syringe - more accurate to measure the hydrogen peroxide than the measuring cylinder because of the differentiation in units. To reduce the errors by 0.1cm³ I decided to use a syringe.
- 50cm³ Measuring Cylinder - this is more accurate to use to measure the volume of oxygen produced rather than counting the amount of bubbles as they vary in size and are occasionally released too fast.
- 15cm³ Beaker-for the hydrogen peroxide
- 500cm³ Beaker-for the water bath
- Clamp Stand – used to make the measuring cylinder stable so I can measure the volume of oxygen produced more accurately.
- 250cm³ Conical Flask - more accurate than a test tube so that the particles can get released more affluent through the delivery tube to the measuring cylinder.
- Stop Watch - I can time the rate of reaction more precisely. Consequently there will be fewer inaccuracies.
Fair Test
In order to make the experiment fair I will be undertaking the following things, so they can be controlled:
- The hydrogen peroxide must be the same throughout the experiment.
- The weight of the kidney beans must be constant.
- Keep the pH constant using pH 7 buffer.
- The time taken to measure the volume of oxygen produced must be constant at 2 minutes, so that I can compare the results of my experiments accurately.
- Time for kidney beans to be grinded must constant, so the surface areas of the kidney beans do not be different as this can affect the enzyme activity.
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The temperature must be kept constant at 25ºC so this does not affect the enzymes activity.
After controlling these variables I would have accurate results when repeating this experiment.
Preliminary Experiments
Before I conducted my experiment I did preliminary experiments just so that I can the right balance of equipment to use. I did two preliminary experiments to give me an indication of what volumes to use.
I soaked for 0 minutes and used 5cm³ of hydrogen peroxide. I grinded the kidney beans for 5 seconds, and added 3 drops of pH 7 buffer. The result I got was that 17.5cm³ of oxygen was produced in a reaction where I used 10g of kidney beans.
This shows that I used less hydrogen peroxide and more kidney beans which in fact made the reaction much slower. Therefore I decided to make some changes in my next preliminary experiment.
Here I soaked for 0 minutes and used 10cm³ of hydrogen peroxide, where I heated this toxic up to 25ºC. I also used a water bath to heat the water to 25ºC. I added 3 drops of pH 7 buffer and used 5g of kidney beans. The amount of oxygen produced was 25.1cm³.
The reaction speeded up quite strongly. The result is therefore more accurate and balanced in my experiment.
Before I had to do the final experiments I decided to make some final changes. I have to alter the timing of the rate of reaction to 2 minutes. Plus I need to have a wider range of soaking times ranging from 0-5 hours. By doing this the experiment ought to be successful.
Diagram
Method
- Set up apparatus as shown above.
- I measured out 5g of the soaked kidney beans on a balance using a weighing boat.
- I grinded the kidney beans for 10 seconds. By doing this I made sure the kidney beans were blended evenly, using a blender.
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I prepared a water bath, which consisted of pouring heated water in a large beaker, at a constant temperature of 25ºC, by using a thermometer.
- After this I put the 5g kidney beans into the conical flask, by transferring the grinded kidney beans onto a sheet of filter paper and into the conical flask.
- Then I added 3 drops of pH 7 buffer.
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Heated 10cm³ of hydrogen peroxide in a beaker, and added this amount into the conical flask, using a 20cm³ syringe.
- I put the bung/delivery tube on the conical flask and timed the reaction using a stopwatch.
- After 2 minutes, I removed the bung from the delivery tube, and measured the volume of oxygen produced in the measuring cylinder. I then recorded the results.
- Repeat steps 2-9 and record the results in a table
- Each experiment was conducted twice so an average could be achieved, as I had carried 6 different experiments with soaking times ranging from 0-5 hours, with 0 representing the beans have not been soaked.
I had made sure that I kept all the variables that I said to keep constant in these experiments, and controlled the variables to keep the experiments fair.
Safety Hazards
These are the dangers that I can come across while doing this experiment, and how to overcome these dangers:
- I will have to make sure I wear goggles for eye protection so no toxic i.e. hydrogen peroxide can harm my eyes.
- I would have to wear a lab coat and gloves for the protection of hands.
- Put the stools in so there are fewer chances of accidents happening.
- Do not leave equipment over the edges of the workplace, hence reducing accidents.
- Handle glassware with care due to its fragility.
Implementing
Table of Results
Analysing
I calculated the volume of gas produced in one minute by one gram of bean tissue by using this formula:
Average
Mass of Beans (gˉ¹) x Time Taken (minˉ¹)
I calculated the average for experiments as follows:
0 Hours – 30.3 30.3
5 x 2 = 10 = 3.03 (cm³gˉ¹minˉ¹)
1 Hours – 39.0 39.0
5 x 2 = 10 = 3.9 (cm³gˉ¹minˉ¹)
2 Hours – 46.0 46.0
5 x 2 = 10 = 4.6 (cm³gˉ¹minˉ¹)
3 Hours – 51.95 51.95
5 x 2 = 10 = 5.195 (cm³gˉ¹minˉ¹)
4 Hours – 52.0 52.0
5 x 2 = 10 = 5.2 (cm³gˉ¹minˉ¹)
5 Hours – 52.45 52.45
5 x 2 = 10 = 5.245 (cm³gˉ¹minˉ¹)
Here are these results included in the results table:
Conclusion
There is a definite trend in my results. As the length of soaking time increases so does the rate of reaction until the rate reaches 4 hours where the rate keeps constant. This is evidently noticeable from my results table and graph.
By analysing my results I notice that my prediction was correct. As the soaking time increases so does the rate of reaction as the process of osmosis is taking place. But there reaches a point where the graph levels off as the cell becomes turgid. This shows that my results do back up my prediction.
Water is needed for catalase activity to take place. A mature seed contains 10% water. This explains how hydrogen peroxide gets decomposed when it goes through no soaking at all. The seed is very stable and activity will surely increase as more water is needed. When the water is added to the bean, this stimulates the seed to germinate into a plant. Protein, lipids and starch are then converted into growing plant stalk and root. These are conversions storage polymers to plant structural proteins and carbohydrates, which is done by the enzyme catalase. The seed only partially germinates.
Lipids stored in the plant are converted to energy and other molecules are a highly oxidative process. The by-product of this lipid-metabolism is hydrogen peroxide. This is why there is an increase in the rate of enzyme activity, as there is high lipid metabolism in a germinating seed.
Metabolic activity occurs in the embryo as water goes into the cells by osmosis. This is the movement of water particles from a region of higher water potential to a region of lower potential through a partially permeable membrane. Inhibition will carry the water through to the microphyle, which is causes the seed to swell and then rupture. The metabolic rate will be nil, so by osmosis the water levels will reach approximately 75% in each cell. Metabolism can then reach its optimum level and let growth begin.
Seed structure determines what goes in and out of the kidney bean. This has a protective layer called the testa. This is where the catalase exists. The catalase can only be activated when the testa breaks apart.
In an enzyme-catalyzed reaction, the substance to be acted upon (the substrate) binds to the active site of the enzyme. The energy is reduced to activate the reaction so the products are formed. Thus the hydrogen peroxide binding to the catalase. This is known as an enzyme-substrate complex. This is how the molecules move inside the cell.
The molecules travel due to Brownian motion.
These enzymes use the lock and key theory to fit into an active site.
This is a simple way of describing how specific an enzyme is for its substrate. Each enzyme is a protein which is a polypeptide chain folded into a complex 3 dimensional structure. Part of that structure contains the active site where the enzyme binds to the substrate to form a chemical reaction.
The graph then levels off as the rate of reaction becomes constant. Reason being, is that water enters the beans through osmosis. In a plant cell a limited number of water can enter the cell because the protoplast shrinks, but the cell wall restricts the cell from bursting. The vacuole is full of water, as water can no longer enter the cell. The cell becomes turgid as the cell is fully inflated with water.
Evaluation
On a whole I think the experiment went quit well as I got similar results and graphs to what I was hoping to get. This enabled me to answer the question I was set.
There are no anomalies in my results which show how well this experiment went.
Errors
- Not using correct apparatus for the measurement of hydrogen peroxide.
- Not reading off the volume of oxygen precisely.
- Not weighing the beans correctly.
- Leaving the beans a bit longer than they should have been soaked for.
Improvements
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Use an accurate syringe to the nearest 0.1cm³.
- Get down to eye level and measure the amount with a paper so I can read off the numbers more accurately.
- Staying at the balance until the final value is shown.
- Timing accurately and starting the experiment as soon as the beans are soaked.
Limitations
I think that I could have used a wider range of soaking times. For example instead of using 0-5 hours I could have done 0-20 hours at intervals of about 4. This might have given me a clearer picture of the results. It would have given me additional knowledge of the rate of reaction.
I could have also timed the experiment for longer to about 4 minutes to see what happens to the gas, and therefore the rate. This method might have given me more accurate results.
I also would have liked to have repeated each experiment at least 3 times. This would enable me to produce better averages, and therefore resulting in more reliable results.
Accuracy
I would say my results are fairly accurate as they do resemble my prediction. By observing my graph I notice that there are two points which are not neatly on the line following the pattern of the graph.
The first point is at 2 hours – 46.0cm³
The second point is at 5 hours – 52.45cm³
I think the main reason as to why these were a bit inaccurate was due to me leaving these beans to soak longer than necessary, as I did not use a stopwatch to time the soaking and instead used a normal watch.
When comparing my results with a friend who had done the same experiment I established that the results were fairly similar. This also proved to me that my experiment was comparatively accurate.
Reliability
My results are reliable as they contradict the ones that are in the text book. They also follow the same results as my friends. This gives me a somewhat idea of how reliable my results are.
From analysing my results I can say that my results my original result are fairly similar to the replicate experiments. This again shows how successful my experiment was, as I had less errors and more accuracy.
I am able to draw a valid conclusion from my results and say that as the soaking of the beans increased so did the rate of reaction, but soon the rate kept constant.
