Catalase: Structure and Function

The enzyme catalase is part of most organisms' defense against the superoxide radical anion, O₂^-, a harmful by-product of the metabolic oxidation of fats and carbohydrates (4). The enzyme superoxide dismutase is the first line of defense against O₂^-. It converts superoxide ion to hydrogen peroxide, which is still quite toxic to cells. Catalase is responsible for converting the hydrogen peroxide to water and oxygen:

In the second reaction depicted above, hydrogen peroxide is both oxidized and reduced. This is thought to be a two-step process, and catalase is involved in both steps (4, 5). In the first step, one molecule of hydrogen peroxide is reduced to water as catalase is oxidized to Complex I. The second molecule of hydrogen peroxide is oxidized in the second step, as Complex I is reduced back to catalase:

The decomposition reaction follows first-order kinetics within short reaction times (<3 min) at relatively high enzyme concentrations (6). In addition to the mechanism described above, catalase is gradually, irreversibly oxidized by hydrogen peroxide, so long reaction periods or dilute solutions of enzyme result in deviation from first-order behavior. Catalase functions best at pH 7, and is denatured in a basic environment above pH 10 (6).

The detailed structure of catalase differs from one organism to another, but the general quaternary structure is analogous to hemoglobin in that catalase is tetrameric and each 500-residue subunit contains an iron-centered porphyrin ring. However, unlike hemoglobin's iron, which is Fe(II), catalase utilizes Fe(III). This iron is formally oxidized to Fe(V) in the oxidation-reduction cycle, although spectroscopic evidence suggests that Complex I is more likely to be an Fe(IV)-porphyrin cation (4).

Experimental Procedure

The catalase project is used in the second semester of our chemistry course for non-science majors. Students typically spend two laboratory periods following a designed procedure from their laboratory handout. They can then explore different aspects of the reaction during one or two additional lab periods.

Catalase extracts are obtained by combining equal amounts (by mass) of plant or animal material and deionized water in a blender, although a food processor or juicer also works well. Alternatively, the potato can be ground up using a mortar and pestle and then mixed with water. The extracts are filtered through cheesecloth. (Glass wool can be substituted.) A gas collection apparatus is prepared using a stoppered test tube or Erlenmeyer flask with a side arm connected by rubber tubing to a graduated cylinder that is filled with water and inverted in a large water-filled beaker. The catalase extract (2-3 mL) is mixed with hydrogen peroxide (3-5 mL of a 3% solution) in the side-armed vessel, and students monitor the catalase activity by measuring the volume of oxygen generated by the reaction as a function of time.

After working out the general procedure using potato extract, the students repeat the experiment several times incorporating the following variations: alternative sources for the enzyme, different concentrations of hydrogen peroxide, higher and lower temperature, different pH, different substrate or catalyst, and addition of an inhibitor. They then design their own experiment to further explore some aspect of this or another enzymatic reaction.

We pursued this process in more detail in our research laboratories. Catalase extract was prepared as described above, with the exception that phosphate buffer solutions (pH 7) were used in place of deionized water. In some cases, the filtered extract was also centrifuged. Generally, a less concentrated solution of hydrogen peroxide was used (<1% w/w). Conditions were selected to favor first-order behavior, which was observed in most cases. More quantitative kinetic measurements were obtained by using a UV-vis method of analysis (6, 7). In this method the disappearance of hydrogen peroxide was determined by monitoring the decrease in absorbance at l = 240 nm, which corresponds to the n -> s* transition in hydrogen peroxide. More consistent UV-vis results were obtained when the catalase extracts were centrifuged.

Results and Discussion

Sources of Catalase

Extracts from different vegetable and animal sources were investigated for their catalase activity. The results are listed below.

The activity of catalase varied significantly from one source to another. Among the plants tested, leeks and parsnips contained the highest activity per mass of plant. We found that fruits typically have much lower catalase activity, perhaps due to their higher acidity. This may result in some fruits having greater susceptibility to damage by oxidation. Quantitative measurements of hydrogen peroxide decomposition were made by UVvis spectroscopy to compare the activity of catalase from some of the sources. These results are summarized in the table below, expressed relative to potato. Measurements for different samples of the same vegetable varied somewhat, presumably due to variations in water content.

pH

Our students were able to obtain results consistent with literature reports that catalase activity decreases by half when the pH is lowered from 7 to 3 (6). The greater acidity of many fruits compared to vegetables could explain the decreased activity of fruit catalase extracts. Acetate and formate are not recommended for pH studies because of their inhibitory effects (see below).

Temperature

Students in the laboratory were instructed to heat a sample of catalase extract to temperatures above 90 °C for a short time and then measure its (diminished) activity at 35 °C. Some students in the open-ended laboratory devised an experiment in which they heated the catalase extract­hydrogen peroxide mixture to temperatures above 90 °C. They expected no reaction owing to the total denaturing of the enzyme. They were surprised to find increased oxygen production, and mistakenly assumed it was due to increased catalase activity. However, they achieved similar production of oxygen when they omitted the catalase solution entirely!

Figure 1. Catalase activity at different temperatures, corrected for the thermal decomposition of H₂O₂. Catalase activity is given as the rate of H₂O₂ disappearance relative to the slowest rate, defined as 1 (at 55 °C), as measured by UV-vis spectroscopy.

This is an excellent example of the advantages of a catalyst in general, and an enzyme in particular. Students can see that the uncatalyzed reaction occurs readily, but only at temperatures that are prohibitive for most living organisms. The enzyme enables this reaction to occur rapidly at physiologic temperatures.

Inhibition

Numerous substances inhibit the function of catalase (8), but many of them are too toxic to be suitable for a teaching laboratory. Relatively safe and readily available catalase inhibitors include acetate, ascorbate, ethanol, formate, methanol, and nitrite. Caution: ethanol and methanol are flammable. The addition of small amounts (10­20 mg per milliter of plant extract) of these inhibitors significantly slows down the catalase-catalyzed decomposition of hydrogen peroxide.

Different Substrate or Catalyst

The decomposition of hydrogen peroxide can also be catalyzed by substances other than catalase: for example, CuCl₂ (2). The relative catalytic ability of catalase can be compared to these other catalysts. Some students also substituted tert-butyl-hydroperoxide for the hydrogen peroxide substrate. Not surprisingly, substituting a different substrate significantly decreased the rate of oxygen production.

Conclusion

We have found this to be an excellent experiment to introduce an inquiry-based project. Students can individually investigate a large variety of reaction features, most of which produce data that can provide insight into the sources and activity of the enzyme catalase and enzyme behavior in general. The project can be approached at different levels of quantitation, depending on whether one expects students to understand kinetics at a quantitative or qualitative level. There are numerous aspects of this reaction that remain unexplored. This, coupled with the fact that the materials and apparatus for the oxygen-collection method of analysis are inexpensive, safe, and readily available, makes this an attractive project for high school students.

Literature Cited

1. Summerlin, L. R.; Borgford, C. L.; Ealy, J. B. Chemical Demonstrations, A Sourcebook for Teachers, 2nd ed.; American Chemical Society: Washington, DC, 1988; Vol. 2, p 152.

2. Roberts, J. L., Jr.; Hollenberg, J. L.; Postma, J. M. General Chemistry in the Laboratory; W. H. Freeman: New York, 1975.

3. Heasley, V.; Christiansen, F.; Heasley, G. Chemistry and Life in the Laboratory: Experiments in General, Organic, and Biological Chemistry, 2nd ed.; Burgess: Minneapolis, 1983.

4. Walsh, C. Enzymatic Reaction Mechanisms; W. H. Freeman: San Francisco, 1979.

5. Murshudov, G. N. FEBS Lett. 1992, 312, 127.

6. Luck, H. Methods of Enzymatic Analysis; Bergmeyer, H. U.; Gawehn, K., Eds.; Academic: New York, 1974; pp 885­894.

7. 1994 Catalog of Biochemicals, Organic Compounds, and Diagnostic Reagents; Sigma Chemical Company: St Louis; "Catalase", p 222.

8. Jain, M. K. Handbook of Enzyme Inhibitors, 1965-1977; Wiley: New York, 1982.