Experimental Question: What effect does substrate concentration have on the rate of enzyme activity?

Experimental Procedure:

1. Work together in groups of 3-4 at a lab table and have one of the members be responsible for timing the other members.
2. Place the test tube in a rack. Fill the test tube with 30mL of 3.0% Hydrogen Peroxide (H2O2) using the 100mL Graduated Cylinder. Do not add any water to this solution.
3. Obtain the 6mm Disks and dip the first disk in the freshly prepared enzyme solution.
4. Place the disk under the surface of the rubber stopper.
5. Insert the stopper into the test tube containing the peroxide solution to form a leak proof reaction chamber.
6. Invert the test tube which would initiate the decomposition of hydrogen peroxide by the enzyme and start the stopwatch.
7. As the enzyme decomposes the hydrogen peroxide into oxygen and water, the oxygen bubbles will be trapped between the fibres of the filter paper disk. After enough time, sufficient oxygen will accumulate to float the disk. When the disk reaches the surface, stop the timer.
8. Record the time in the observations table.
9. Repeat the procedure thrice for better results and record the time in the observations table.  
10. Next, fill the test tube with 20mL of Hydrogen Peroxide (H2O2) and dilute it with 10mL of Distilled Water using the 100mL Graduated Cylinder. This is the 2% concentrated substrate solution.
11. Repeat the procedure from steps 3 to 7 by performing in total of 4 trials and record the time in the observations table.
12. Again, fill the test tube with 10mL of Hydrogen Peroxide (H2O2) and dilute it with 20mL of Distilled Water using the 100mL Graduated Cylinder. This is the 1% concentrated substrate solution.
13. Repeat the procedure from steps 3 to 7 by performing in total of 4 trials and record the time in the observations table.
14. Finally, fill the test tube with 30mL of Distilled Water using the 100mL Graduated Cylinder. Do not add any Hydrogen Peroxide to this solution. 
15. Repeat the procedure from steps 3 to 7 by performing in total of 4 trials.
16. If there was no reaction after a minute, record time more than 30s in the observations table.
17. Clean up the work area and return all the materials to their allocated places.
18. After the experiment, each members of the group will calculate the average time for the enzyme activity and construct a bar graph of their averages. The four different substrate concentrations should be on the X-axis and the average time should be on the Y-axis.

Observations:

Table 1. Qualitative Observations for Different Types of Substrate Concentrations

Table 2. Rate of Enzyme Activity Recorded Using Different Types of Substrate Concentrations at 4 Trials

Analysis:

Sample Calculation:

Calculation for Average Rate of Enzyme Activity:      ∑ trials

                                                                4

                                                          = 4.21s + 7.39s + 2.38s + 4.12s                                                                                                     4

                     Average Rate of Enzyme Activity = 4.525s 

Table 3. Summary of Average Rate of Enzyme Activity by Four Different Types of Substrate Concentrations each at 4 different trials

Conclusion: 

This experiment was performed for the purpose of investigating the effect of substrate concentration over the enzyme activity. The rate of enzyme activity of catalase obtained from beef liver is examined by using hydrogen peroxide as its substrate. The assay system used in this lab consists of collecting gas produce when the catalase enzyme is mixed with H2O2. Therefore, the assay technique was effective in determining the presence, absence, quantity or rate of change of a substance. In conclusion, as the substrate concentration decreases the rate of enzyme activity decreases. This conclusion is supported by the prediction and by the evidence gathered in Table 3. The rate of enzyme activity becomes highest when the concentration of enzyme is highest. With higher concentration of enzyme, there is a greater chance of effective collisions between the enzyme and H2O2 molecule. As the number of substrate molecules decreases, the number of collisions between the enzyme and the substrate decreases. Therefore, the more substrate molecules are available, the more frequently there is access of active sites of enzyme molecules.

Evaluation: 

There were many sources of errors in this experiment. In the test tube, we found that in one area if there is less substrates, there is less probability to enter in the active site, whereas more substrates will increase the probability to enter in the active site. The faster the reaction, the more hydrogen peroxide decomposed. Therefore, the rate of reaction increases as substrate concentration increases. But there must be a limit to how much substrate is available. At the limit point, when the product is immediately produced, the next substrate should put into the active site and this should go on until the substrate is gone. The only thing that may stop the enzyme activity of saturated substrates would be allosteric inhibition and feedback inhibition. This means that the enzyme will always work until all the substrate is decomposed even though saturated.

        Having more trials with more concentration of Hydrogen Peroxide would have improved the results. It would help         

        The volumes of the liver extract are slightly different from each other and this may affect the experiment’s results. The volume of the hydrogen peroxide might be changed because it is very reactive and some of the substrate has already broken into water and oxygen. This will somehow influence the experiment results.