Enzyme Lab – Measuring the Effect of Substrate Concentration

Enzymes are proteins that serve as biological catalysts in a wide variety of life-sustaining chemical reaction that take place in cells. As catalysts, enzymes lower the activation energy (the amount of energy required to make a reaction occur). By lowering the activation energy, ezymes serve to speed up the rate at which reactions occur.

Enzymes are said to be substrate-specific. A substrate is a molecule that temporarily binds with the enzyme at an area on the enzyme called the active site. Each enzymes catalyzes one specific reaction because there is only one type of substrate molecule with the exact shape that will fit into the enzyme’s active site.

For example, the enzyme amylase will only act on the starch called amylose. The enzyme sucrasewill only act on the sugar called sucrose because it is the only substrate that can fit into the active site of the sucrase enzyme. The substrate is then converted into its products, and the enzyme is free to repeat the process with another substrate molecule (Figure 1).

Figure 1

Your cells and the cells of most living organisms contain an enzyme called catalase. Cells use the enzyme catalase to break down hydrogen peroxide, H2O2, a poisonous byproduct of cell reactions. You have probably seen evidence of this reaction if you have ever poured hydrogen peroxide on a cut. The catalase decomposes hydrogen peroxide into water and oxygen. The oxygen gas is released as bubble. The rate at which this occurs depends on the number of catalase and hydrogen peroxide molecules.

Procedure:

  1. In the conclusion questions section, write an “if-then” hypothesis statement that answer the following question: What effect does increasing the concentration of hydrogen peroxide have on the rate of decomposition by catalase?
  2. Observe your teacher perform a trial using 100% hydrogen peroxide. Use the result of this trial as “trial 1” for 100% peroxide in the data table below.
  3. Read the introduction to the lab while your teacher passes out the catalase solution.
  4. Now observe your teacher perform a trial using 100% hydrogen peroxide with a catalase solution that has been boiled. You don’t have to put this information into the data table, but you will have a conclusion question asking about this scenario.
  5. Now go to your lab station to perform the experiment. Note: If any disk takes longer than 180 seconds to float to the surface, simply record this time as 180+ in the data table.
  6. Do this first trial as a group of 4 to make sure everyone knows how to properly run the experiment. Using a graduated cylinder, measure out 20 mL of hydrogen peroxide. Pour this amount into a small beaker.
  7. Using forceps, pick up one paper disk and submerge it in the enzyme solution for 5 second. Do not let go of the disk.
  8. Remove the disk from the solution and use a paper towel to blot off the forceps and disk to the best of your ability.
  9. Use the forceps to place the disk on the bottom of the beaker with the hydrogen peroxide. Begin timing as soon as the disk touches the surface of the hydrogen peroxide.
  10. Record the time required for the disk to begin floating to the surface of the hydrogen peroxide (stop your timer once the disk is completely off of the bottom of the beaker).
  11. For the rest of the trials, each group of shoulder partners will perform 1 of the 2 trials, and you will then average your results as a team. Repeat steps 6-10using 80%, 60%, 40%, 20%, and 0% hydrogen peroxide solution by diluting the peroxide with water. Use new material (peroxide, disk, water, etc.) every time. After each trial, take out disk with forceps, place on a paper towel, pour peroxide down drain, rinse out beaker, and move on to next trial.
  12. 80% peroxide  16 mL peroxide, 4 mL water.
  13. 60% peroxide  12 mL peroxide, 8 mL water.
  14. 40% peroxide  8 mL peroxide, 12 mL water.
  15. 20% peroxide  4 mL peroxide, 16 mL water.
  16. 0% peroxide  0 mL peroxide, 20 mL water.
  17. Perform 1 final trial using 100% peroxide, but this time put 5 mL of hydroxylamine hydrochloride as well into the beaker as well.
  18. Record your team’s data on the data table on the board so we can find the class averages.

Data and Observations

Percent Hydrogen Peroxide / Time (s)
Trial 1 / Trial 2 / Average
100
80
60
40
20
0
100% plus hydroxylamine hydrochloride
(only need to do 1 trial)

Graph

Make a line graph for the CLASS AVERAGES.To do this, you will need to determine the following:

A) The independent variable ______

B) The dependent variable ______

Conclusion Questions

Hypothesis Statement:

  1. For this experiment, what was the substrate, and what was the enzyme?
  1. What causes the disks to float to the surface?
  1. Which concentration of catalase had the shortest reaction time and why?
  1. Which concentration of catalase had the longest reaction time and why?
  1. Based on the graph and the overall slope of the line, what can you conclude about the effect of substrate concentration on reaction time?
  1. How would the results be different if you repeated the experiment using a different enzyme besides catalase? Explain your answer.
  1. Describe the effect hydroxylamine hydrochloride has on the reaction time.
  1. What type of molecule is hydroxylamine hydrochloride?
  1. A student forgets to dry the tip of the forceps after dipping the disk in catalase solution. What effect will this error have on the reaction time for that trial? Explain.
  1. Explain what would happen to catalase in the following situations, and what would subsequently happen to the reaction rate because of this scenario.
  2. Extremely cold:
  1. Extremely hot:
  1. Extremely basic or acidic:
  1. Extremely high salt concentrations

Pre-AP Enzyme Lab – Free Energy Extensions

  1. Explain the 1st law of thermodynamics.
  1. Explain the 2nd law of thermodynamics.
  1. All chemical reactions involved a conversion of energy. The positioning and movement of what subatomic particle is responsible for this energy?
  2. Was the reaction observed in today’s lab an example of dehydration or hydrolysis? Explain.
  1. Was the reaction observed in today’s lab an example of anabolism or catabolism? Explain.
  1. Was the reaction observed in today’s lab endergonic or exergonic? Explain.
  1. Was the reaction observed in today’s lab spontaneous or nonspontaneous? Explain.
  1. Explain what is meant by each of the variables in the following equation: G = H – T S
  1. Explain the following graph and what each letter represents.