Rate of enzymes reaction and the concentration of substrate
Rate of enzymes reaction and the concentration of substrate
Aim to investigate the effect of substrate concentration on rate of enzyme reaction, using immobilised catalase.
Hypothesis the rate of reaction will be increasing until the limited amount of enzymes. After the point of limit, the reaction rate will be stop increasing and stay at the certain point.
Apparatus
Syringes
Various sized beakers
Stop clock
Manometer
Distilled water
Tweezers
6 boiling tubes
Pieces of liver
Homogeniser
Buffer solution (PH7)
Test tube rack
Sodium alginate solution (2%)
Blender
Calcium chloride solution (1.5%)
Hydrogen peroxide (20 volume)
Ruler
Glass rods
Marker
Risk assessment :
. A hot water bath was used, special attention should be paid.
2. hydrogen peroxide solution was used, it should be handled with care as it is irritant.
3. Safety glasses should be worn for eye protection.
4. Enzymes are biologically active proteins, it should be handled with care. We should avoid direct contact or inhalation.
5. If we spill any peroxide solution or enzyme, spillages should be cleaned up immediately. We should not allow them to dry and generate dust.
Precaution
. Instruction should be read carefully before starting the experiment. We should know clearly that we have to vary the concentration of substrate and investigate the reaction of different concentration of amylase with a fixed amount and concentration of enzyme.
2. All test tubes should be labelled according to their contents.
3. We should work on a concentration at a time to avoid confusion.
4. Separate pipette and syringe should be used for that particular concentration to avoid contaminating.
5. solutions should be made with precision measurements of solute and solvent and well-mixed before use.
6. during the reaction, beads should not be located on the surface. Hence we have to swirl around the test tube well.
Method
Preparation of catalase immobilised in alginate
. chop the liver into pieces and place them homogenise with 50cm . pH7 buffer solution.
2. take 4 cm of the homogenate. And mix them thoroughly with 16 cm of 2% sodium alginate solution. Draw the mixture into 20 cm syringe.
3. Fill the beaker with 200 cm 1.5% calcium chloride solution. Gently push down on the syringe plunger, so that small evenly sized drops of the mixture in the syringe fall into the beaker of calcium chloride. They will set to form alginate beads, with catalase molecules immobilised inside the gel.
4. Allow the beads to harden for 5 min. then carefully, pour off the calcium chloride, and rinse with several changes of distilled water, taking care not to lose the beads. The immobilised catalase is now ready for use.
Main experiment (Rate of enzyme reaction investigated)
. Prepare 20 volume hydrogen peroxide, whose concentration is 1.78 mol/dm .
2. Prepare dilutions of this solution using the following table.
Volume of H O
Volume of distilled water
Concentration of mixture
5 cm
5 cm
.78M
0 cm
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4. Allow the beads to harden for 5 min. then carefully, pour off the calcium chloride, and rinse with several changes of distilled water, taking care not to lose the beads. The immobilised catalase is now ready for use.
Main experiment (Rate of enzyme reaction investigated)
. Prepare 20 volume hydrogen peroxide, whose concentration is 1.78 mol/dm .
2. Prepare dilutions of this solution using the following table.
Volume of H O
Volume of distilled water
Concentration of mixture
5 cm
5 cm
.78M
0 cm
0 cm
0.89M
5 cm
5 cm
0.44M
2 cm
8 cm
0.178M
Keep the diluted substrate in labelled beakers.
Mix well with a grass rod to obtain homogenised solution during dilution. The amount of solution and water can be variously used.
3. place 25 catalase beads in each boiling tubes.
4. Use syringes to measure 10 cm of undiluted peroxide into q boiling tube. Seal the tube with manometer straightaway and mark the original starting point. Allow the reaction to take place for 1 min. Give the boiling tube continuous swirls to prevent the beads staying on the surface.
5. After 1 min, take the final measurement on the manometer. Obtain the length between the starting point and the end point. (also take account the bubbles on the other part of manometer for accuracy.) This is the volume of oxygen formed as a product.
6. Also measure 20 cm of other diluted solutions and distilled water into boiling tubes and label the properly. And do the same procedure as shown above (step 4 and 5)
Alternative method to get the result 1
(STARTS FROM STEP 2 OF MAIN EXPERIMENT)
3. use syringe to measure 10 cm of undiluted peroxide into a test tube. Add 1 drop of neat detergent to stabilise the form, and mix well without causing frothing. Now place 5 immobilised beads in the tube. And time how long the form takes to reach the top of the tube. (this is a simple method of measuring the volume of oxygen produced, assuming non escapes by bubbles bursting). Record this time, and measure the height of form column with a ruler.
Alternative method to get the result 2
(START FROM STEP 2 OF MAIN EXPERIMENT)
3. Place 20 beads of enzyme into the conical flask which is in the water bath which is at room temperature (this is the coolant for the exothermic reaction.)
4. Use syringes to measure 20 cm of undiluted peroxide and pill a conical flask.
5. Seal the conical flask with this devised cork
immediately which the end of the rubber tube
connected to the gas syringe to measure the gas
generated.
6. Let the reaction continue for 10 min and record the result.
7. Repeat the identical experiments to the other solutions.
Result
The result I obtained is shown below.
Conc. of solution (M)
Oxygen produced(cm ) 1st
Oxygen produced(cm ) 2nd
Average amount of oxygen produced(cm )
0
0
0
0
0.178
2
0
1
0.44
36
31
33.5
0.89
84
78
81
.34
11
07
09
.78
55
63
59
As the graph attached, we can see the graph is almost a straight line and the concentration of substrate is directly proportional to the rate of reaction.
Analysis
Enzymes are highly specific. Each enzyme will only catalyse one reaction. The substance with which the enzyme( in this experiment the enzyme was catalase in peroxisomes) combines is known as the substrate (hydrogen peroxide is the substrate in this experiment), which combines with the enzyme at a particular place on the enzyme's surface called the active site.
Since hydrogen peroxide is the by-product of the metabolism reaction, which is a potentially very harmful oxidising agent, our body have to decompose it into harmless water and oxygen by using catalase.
The reaction is;
2H2O2 (aq) ? 2H2O (l) + O2 (g)
catalase
Enzyme, as being proteins, are sensitive to changes in their environment. Changes in concentration of substrate will affect the rate of an enzyme-catalysed reaction.
In this experiment, clearly, when the concentration of peroxide increases, the rate of reaction increases. When the concentration of peroxide increases, there are more peroxide molecules, so the chance of collision between enzyme molecule and substrate molecules also increases. Therefore, more substrates could combine with the active sites to create a new energy profile, for the reaction, with a lower free energy of activation. This speeds up the reaction since enzymes increase the rate of reactions by reducing the free energy of activation. When more substrate molecules are held by the active sites of amylase, the rate of formation of enzyme-substrate complexes increases, the faster the hydrogen peroxide are broken down into products, which are water and oxygen.
The reason the enzymes should be immobilised is that
. enzyme can react thoroughly with substrate.
2. enzyme can be used many times due to the fact enzyme is bound in an agitate.
3. the product is enzyme-free.
4. the stability of enzyme is increased by this.
5. enzymes can be used over and over so it is beneficial to the industry.
Limitation
* Using manometer is not precise enough.
When we set the apparatus to start with, we seal with the manometer. the problem is when we push down the cork, manometer's degree is suddenly change and it may cause the impossibility to measure further. ( the gas was excessively evolved so that the degree of solution exceeds the limit of manometer.) moreover, it is not very accurate to measure with the ruler.
And when we swirl around the test tube, it causes unwanted bubble gab in the tube of manometer which results in the in accuracy of the experiment.
At the very end of experiment, we need to read the result from the manometer at exact limited time. But when we are obtaining the result, manometer's degree keeps arising due to the continuing reaction. Despite the mark we notified, it is still inexact.
Likewise, the diameter of glass tube used in manometer can be different. This also affect on the result.
The solution for these is using gas syringe with increased volumes of substrate and enzyme (we can obtain enlarged amount of oxygen that we can measure by gas syringe.) instead of using a manometer.
* There wasn't any stagnant point which didn't obey the theory.
In theory, there should be the maximum point of reaction which all the active sites of enzyme are occupied with substrate molecules so that the rate of reaction stops increasing. In my experiment, it seems that there is not any point like that.
This should be due to the excess of enzyme. Using less amount of enzyme beads will help this problem.
* Time was limited in the lesson.
Most of the girls were not able to finish the experiment on time since the process of this experiment took considerable amount of time that expected. we had no time to repeat the experiment and take the average time taken, the data might be less accurate.
The most certain way to gain the accurate result is repeating the experiment and get the average of the data. Therefore, we should repeat the experiments as much as we can.
* Using syringe to make the solution was not precise.
At the beginning of the experiment, we made up a 20 cm of a particular concentration of peroxide solutions . However, we have to use the syringe which we have to think and calculate to made up the acquired volume such as 18 cm (use 10cm syringe and the 5 cm of syringe, finally 3 cm of syringe.) The volume in one test tube might not be exactly the same as the amount that required. Therefore, this also make the data less accurate.
Using pipette is more accurate.
* The number of immobilised enzymes in each beads and the size of beads can be different from each other.
Conclusion
From this experiment, we could conclude that as the concentration of substrate increases, provided that there is an excess of enzyme molecules, the rate of reaction increases in proportional to the concentration of enzymes.
Further work
We could also investigate how the enzyme concentration affects the rate of reaction. We could vary the enzyme concentration, but keep the concentration of substrate molecules constant. Since the rate of the reaction depends on the rate of formation of enzyme-substrate complexes, the rate of reaction will increases as the enzyme concentration increases, but only until all substrate molecules are being used. This shows that both enzyme and substrate concentration affect the rate of reaction.
And as there was no point where all enzyme molecules are occupied (saturated), we can increase the concentration of hydrogen peroxide to investigate the maximum point of catalase for hydrogen peroxide.
