Overview of the Experiment
In this experiment, the student will investigate the enzyme activity of catalase by studying the decomposition of H2O2 to form water and oxygen. Using an oxygen based pressure sensor the student measures the amount of oxygen produced and then calculates the rate of the enzyme catalyzed reaction under various conditions. The student also completes a Protein Assay on an unknown sample of catalase using the Coomassie® Plus Protein Assay from Pierce to determine the protein concentration of the sample. The correlation between the actual and calculated concentration gives an indication of the experimental skills used in carrying out the experiment.
This is an integrated experiment that includes topics from physical chemistry and biochemistry. It is designed to introduce students to the basics of:
- Studying the effects of reaction environment, including temperature, and varying substrate concentration on the rate of an enzyme-catalyzed reaction.
- Combining physical chemistry and biochemistry, principles and theory, with the goal of determining various biochemical and biophysical constants for the enzyme catalase.
- How to acquire experimental kinetic data for an enzyme catalyzed reaction.
- Learning how to perform data manipulation in order to extract out information such as rate constants from experimental kinetic data.
- Correct handling of UV-VIS Spectroscopy
- Operating an Eppendorf Centrifuge and a Pipettman
- Preparation of standard solutions
1 This experiment was synthesized by John J. Dolhun by synthesizing various contributions from course textbooks, current literature, and others affiliated with course 5.310 and 5.301, and updated again on (May, 2017).
2 Apparatus adapted and modified from: Lewis, M. E.; Levine, R. M.; York, J. T.; Grubbs, W. T., Journal of Chemical Education, (2009) 86, 1227-1230.
3 Adapted from: Massachusetts Institute of Technology, Department of Chemistry, 5.301 Chemistry Laboratory Techniques, Protein Assays and Error Analysis, 2009.