Effect of Substrate Concentration on Rate of Reaction

Aim:
The aim of the investigation was to investigate the effect of substrate concentration Hydrogen Peroxide H O (in %) on the rate of reaction of the enzyme catalase (in 1/mean time). Prediction: As the substrate concentration (hydrogen peroxide) in % increases the rate of reaction in 1/mean rate increases until the solution becomes saturated with the substrate hydrogen peroxide. When this saturation point is reached, then adding extra substrate will make no difference. The rate steadily increases when more substrate is added because more of the active sites of the enzyme are being used due to more collisions between the substrate and enzyme because there are more molecules of hydrogen peroxide, so there will be an increase in enzyme-substrate complexes formed which results in more reactions so the required amount of oxygen is made more quickly. Once the amount of substrate molecules added exceeds the number of active sites available then the rate of reaction will no longer increase, as the maximum number of reactions has already taken place. This means that any extra substrate molecules will not to bind to the active site to form E-S complexes as there are limited active sites, in which by this point all the active sites would have been used up.

Experimental Design –
Independent Variable:
The independent variable, is the variable that is changed, this was the substrate concentration in potato discs. The dilutions of Hydrogen Peroxide were 0.2%, 0.4%, 0.8%, 1.2%, 1.6% and 2.0%. Dependent Variable:

The dependent variable is the variable that is measured, in this experiment the dependent variable was time taken for disc to reach the surface. Rate = 1/mean time. Controlled Variables:
The controlled variables were volume of hydrogen peroxide which was 10cm³, concentration of hydrogen peroxide of 2% stock concentration. Also same size discs of the potato which were 2mm. The enzyme concentration has to be kept constant because if more enzymes are present, more active sites will be available for the substrate to combine with it. Same source of enzyme, the potato must be cut out from the same one because there are different amount of catalase present in different sources. Repeatability:

The more repeatable the experiment is the less variation there is and the greater confidence in the mean. Every concentration of the hydrogen peroxide (Substrate) was repeated at least 4 times, whereas some of the concentrations were repeated more times. For example concentration 0.8% and 1.2% were both repeated 13 times. A mean was calculated for each hydrogen peroxide concentration (substrate), which made the results more reliable. Having more repeats, decreases the variance in results therefore making them more reliable. Control Experiment:

The control experiment is using a denatured enzyme by boiling it. This would then be compared to the standard experiment above with an enzyme and different substrate concentrations. For this control experiment, the independent variable would remain the same which is the substrate concentrations. So in this experiment, we would investigate the effect of substrate concentrations on a denature enzyme. The controlled variables would remain the same, so this means that the volume of hydrogen peroxide which was 10cm³ and concentration of hydrogen peroxide of 2% stock concentration would be kept constant. Also same size discs of the potato which were 2mm would stay the same. Safety Risk Assessment:

The major hazard was spilling the Hydrogen Peroxide, because it is irritant. This means when inhaling it can damage lungs and throat causing shortness of breath and coughing. It can burn the eyes and skin and possibly cause eye g. There in order to prevent the spillage of hydrogen peroxide wear a lab coat to avoid any going on the skin or clothes. Wear safety goggles to prevent any contact with the eyes. Furthermore, wear gloves so that no hydrogen peroxide spills on your hands. Another major hazard in this experiment was using the scalpel and cork borer to cut the potato into discs of the right size (of 2mm), you had to be careful with these apparatus because they are sharp and therefore they can cut easily. To prevent this, use the scalpel and cork borer away from your body, and watch careful what you are cutting
so that you don’t mistakenly cut your hands or fingers. However, there are other standard precautions that need to take place such as tying hair back.

Analysis of Results –
Trend:
The overall trend represented by the graph suggests that as the concentration of the substrate (hydrogen peroxide) increases there is an increase in the amount of the rate of reaction of the enzyme catalase. This is because there are more substrate molecules available, so an increase chance of a successful collisions. At 0.2% for substrate concentration (hydrogen peroxide) the rate of reaction for the potato disc to reach to the surface was 0.00295 1/s. At 1.6% of substrate concentration (hydrogen peroxide) the rate for the potato disc to rise to the surface was 0.02922 1/s. Consistency:

From the table of results and the graph, it is clear that there is overlapping within the range bars between hydrogen peroxide (substrate) concentration. This shows that overlapping on the range bars, in the same direction has decreased the confidence of the mean. As the range bars are large, the variance is large so the confidence in the mean has decreased. Sources of uncertainty plus improvement:

There were a number of errors/sources of uncertainty with this experiment, for example the size of the potato discs could have been different sizes in uniform size. In order to improve this, instead of measuring the size of the disc to be 2mm with a ruler use a vernier calliper, this instrument is much more precise to measure small distances such as the 2mm for the potato disc. Another uncertainty from this experiment t was the mass of the potato disc; the mass was not measured so some of the potato discs may have weighed more than others which may affect the results as the potato discs which had a greater mass could have had a greater enzyme (catalase). In order to improve this, instead measure the mass of the potato disc to see if the difference between the masses of potatoes is so great or not.

In addition the concentration of the enzyme could have varied for each potato disc, in order to improve this make the enzyme concentration a control variable. Furthermore, a product of the reaction was oxygen being produced which was shown as bubbles. These bubbles made the potato discs float to the surface. However, not all the discs floated to the surface of the solution; even though oxygen was produced some of the potato discs only summersaulted and then sunk to the bottom of the solution again while oxygen bubbles were still being produced. In order to improve this error, instead of measuring the time taken for the potato disc to rise to the surface, there should be a given time for example 5minutes, to see if the potato disc rises to the top or not. Explain/relate results to theory:

At low substrate concentration, enzyme activity is proportional to substrate concentration .This is because random collision between substrate and active site happen more frequently with higher substrate concentrations. The rate of reaction for potato disc to reach the surface at 0.2% was 0.00295 1/s. At high substrate concentrations, all the active sites of the enzyme are fully occupied, so raising the substrate concentration has no effect. At the substrate concentration 1.6%, the rate of reaction for the potato disc to reach the surface was 0.02922 1/s. The reason for the increase in rate of reaction is because the numbers of substrates are increasing and so there are more to bind to the active site of the enzyme to form enzyme-substrate complexes. However, the increase on the graph slows down between 0.8% and 1.2%. The reason for this may be that due to an error or another variable the enzymes were limited here and therefore there were not enough active sites to bind to the substrate to form enzyme substrate complexes.

Conclusion:
Overall, as the x axis (substrate concentration in %) increases, the y axis (rate of reaction for the potato disc to reach the surface in 1/s) increases, which means that there are more frequent collisions between the substrate and the active site of the enzyme therefore more enzyme substrate complexes are being formed. This is show on the graph as; at 0.4% for substrate concentration (hydrogen peroxide) the rate of reaction for the potato disc to reach to the surface was 0.00613 1/s. At 2.0% of substrate concentration (hydrogen peroxide) the rate for the potato disc to rise to the surface was 0.04000 1/s.

Plan for Further work –
Further investigation with different independent variables:
1. Instead of using potato discs use liver the liver will either be re used liver or fresh liver and crushed liver or uncrushed liver. 2. Change the size of the potato discs to 2mm, 4mm, 6mm, 8mm and 10mm. 3. Temperature could be an independent variable in ºC. The range could be 10 ºC, 20, 30 ºC, 40 ºC 50 ºC and 60 ºC. Controlled variables:

1. I would control the temperature using a water bath; the temperature would be constant at 40ºC because enzymes work best at this temperature (optimum temperature for most enzymes). Along with the controlled variables which would remain the same, so this means that the volume of hydrogen peroxide which was 10cm³ and concentration of hydrogen peroxide of 2% stock concentration would be kept constant. Also same size discs of the potato which were 2mm would stay the same. 2. The controlled variables would remain the same, so this means that the volume of hydrogen peroxide which was 10cm³ and concentration of hydrogen peroxide of 2% stock concentration would be kept constant. I would control the temperature using a water bath; the temperature would be constant at 40ºC because enzymes work best at this temperature (optimum temperature for most enzymes). The substrate concentration of 2.0% would remain the same. 3. Along with the controlled variables which would remain the same, so this means that the volume of hydrogen peroxide which was 10cm³ and concentration of hydrogen peroxide of 2% stock concentration would be kept constant. Also same size discs of the potato which were 2mm would stay the same. The substrate concentration of 2.0% would remain the same. Expected Results:

1. The tube with fresh liver will react faster than the reused liver, the tube with fresh hydrogen peroxide will react faster than the tube with reused hydrogen peroxide, and the crushed liver will react faster than the uncrushed liver. Liver has higher protein content than potato, so the reaction will be faster with the liver. 2. The expected results of using different potato discs is that the larger the potato disc the longer the rate of reaction for the potato disc to reach to the surface as it will require a lot more oxygen bubbles to be produced so the reaction will be slower. 3. The results would be the rate of reaction would increase till 40 ºC which is the optimum temperature for most enzymes. It increases as there are greater collisions between the active site in the enzyme and the substrate and therefore more enzyme substrate complexes are forming. After this temperature, the rate of reaction would decrease as the maximum amount of E-S complexes have been made, now the shape changes and the hydrogen bonds, ionic bonds and covalent bonds start to break so the shape which is maintained by these bonds also break down causing the enzyme to denature.