Title: The effect of enzyme concentration, temperature, pH, substrate concentration, and ionic concentration on the activity of catalase

Abstract The effects of various conditions on the activity of yeast catalase were studied using a simple set up. Increasing enzyme and substrate concentrations increased the rate of oxygen production, whereas increasing NaCl concentration decreased it. Temperature appeared optimal at 37C and 100C stopped the reaction. A pH of 7 resulted in better activity compared to pH 4 or 10. These results are consistent with the general properties of enzymes. Introduction Enzymes like catalase are biological catalysts that accelerate chemical reactions under physiological conditions. Catalase protects cells against superoxide free radicals, by-products of aerobic respiration that can attack proteins, cell membranes, and nucleic acids to cause cellular damage. Another enzyme, superoxide dismutase, converts the free radicals to hydrogen peroxide, and catalase converts the latter to harmless oxygen and water. One molecule of catalase decomposes as many as 40 million molecules of hydrogen peroxide to water and oxygen per second (Goodsell 2004). Reaction rate varies according to the physiological conditions in the cell. These conditions are quite narrow. Like all enzymes, catalase is a protein whose function depends on its three-dimensional configuration or shape, which must be such as to ensure that substrates bind to the enzyme in the proper orientations for reactions to occur. Shape tends to change when molecular interactions, such as atomic distances and bond strengths, between the protein s amino acids change as a result of pH and temperature. Each species has evolved a slightly different kind of catalase that fits its living conditions. Human catalase, for instance, has an optimal pH range that includes pH 7 and an optimal temperature that includes 37C. The activity of catalase and how it is affected by various conditions is easy to study using simple equipment. Hydrogen peroxide is reacted with a catalase solution and the volume of oxygen that is generated is measured. The rate of oxygen production is maximal early in the reaction when substrate concentration is high, then slows down to zero when the substrate is completely consumed. The rate of oxygen production, calculated by taking the difference of cumulative oxygen volume between two time points in the initial phase, is then used as a measure of activity. The pH, temperature, ionic strength, and concentrations of enzyme and substrate can be readily manipulated and their effects on activity compared. The effects of enzyme concentration, temperature, pH, substrate concentration, and ionic concentration on the activity of catalase were studied in this experiment. Results showed that all affect activity. Methods The procedure was taken from www.explorebiology.com/documents/LabEnzymeCatalysis2007.pdf. Briefly, a reaction vessel was sealed with a stopper into which was inserted a tube that connected the reaction vessel to a graduated cylinder filled with water in such a way that gas from the reaction vessel would enter the cylinder and form an air bubble whose volume can be measured. This cylinder was immersed with its mouth down in a pan of tap water. The reaction vessel contained 10 mL of 3% hydrogen peroxide in distilled water at the same temperature as the water in the pan. Upon addition of 1.0 mL of yeasts catalase solution, the reaction vessel was immediately stoppered and immersed in the pan. The volume of the oxygen air bubble in the graduated cylinder was recorded over a span of 5 minutes at intervals of 30 sec. A baseline activity at room temperature was taken using this set up.

The experiment was then repeated with various changes to study the effects of reaction conditions. To test the effects of enzyme concentration, rather than 1.0 mL of catalase, 0.75, 0.50, and 0.25 mL were used instead. To test for the effect of temperature, the water temperatures in the pan and the reaction vessel were equilibrated for 5 minutes at 5, 37, and 100C before the experiment was run at these temperatures, except for the 100C set-up where only the reaction vessel was boiled. To test for the effects of pH, 10 mL solutions of hydrogen peroxide were made by adding 5 mL of pH buffers at pH 4, 7, and 10 to 5 mL of a 3% solution of hydrogen peroxide. To test for the effects of substrate concentration, 10 mL solutions of 3%, 1.5%, 0.730% and 0% hydrogen peroxide were prepared from the stock 3% hydrogen peroxide solution using distilled water as the diluent. To test for the effect of ionic concentration 1.5% hydrogen peroxide solutions were prepared containing final concentrations of 0%, 1%, and 5% of sodium chloride. The 5% sodium chloride solution was prepared by adding 5g of NaCl to 50mL of distilled water and then adding 5 mL of this to 5 mL of a 3% hydrogen peroxide solution. Other concentrations were prepared accordingly.

Figure 2.Volume of oxygen produced by the catalase reaction as a function of time and enzyme concentration. Reaction contained 0.75 to 0.25 mL catalase solution, 10 mL hydrogen peroxide (3%), at room temperature.

Varying Concentrations (Part B)

10 9

7 mL of H20 displaced

6 0.25mL catalase 0.50mL catalase 4 0.75mL catalase 1.00mL catalase 3

0 0 30 60 90 120 150 seconds 180 210 240 270 300

Figure 3.Volume of oxygen produced by the catalase reaction as a function of time and temperature. Reaction contained 1 mL catalase solution, 10 mL hydrogen peroxide (3%), at 5C, 37C, and 100C.

Varying Temperature (Part C)

mL of H 2O displaced

Time in seconds

Varying pH (Part D)

mL H2 O displaced

pH

pH pH

Time in seconds

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Figure 5.Volume of oxygen produced by the catalase reaction as a function of time and substrate concentration. Reaction contained 1 mL catalase solution, 10 mL hydrogen peroxide (3%, 1.5%, 0.3%, and 0%), at room temperature.

Varying substrate concentration (Part E)

10 9

mL H2 O dis laced

0.0% ubstrate 0.30% substrate 1.50% substrate 3.00% ubstrate

0 0 30 60 90 1 0 150 Time in seconds 180 10 0

300

42

32

4
6

Figure . Volume of oxygen produced by the catalase reaction as a function of time and NaCl concentration. Reaction contained 1 mL catalase solution, 10 mL hydrogen peroxide (1.5%), NaCl (5%, 1%, or 0%), at room temperature. The 0% data was taken from the pH 7 data (Figure 4).
7

Varying ion concentration (Part F)

4.5

mL H2 O dis laced

.5

1.5

0.5

0 0 0 0 90 1 0 150 Time in seconds 180 10 40 70

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Discussion The graphs show that oxygen production increases with time at rates that vary according to the reaction conditions. The rate is faster with higher enzyme concentration (Figure ) and with substrate concentration (Figure 5). This is expected of catalysts. The rate is faster at 7C as compared to 5C or 100C (Figure ), suggesting that 7C is optimal. At 100C the reaction stopped, and this may be because the enzyme had been denatured, that is, misshapen to the point of being non-functional. The effect of pH was best at pH 7 (Figure 4), which is expected since the yeast from which the catalase was obtained thrives at around normal pH rather than in extremely acid of alkaline environments. Salt concentration appears to have lessened the activity (Figure ). This is not surprising given that the optimal physiological NaCl concentration for yeast is between 0.04 and 0.0 % (Lamontagne et al. 000). In all cases, the rate appears to have been most rapid during the first 0 seconds, then began slowing down after that. Figure 7 shows how much oxygen was produced at every 0 second interval for the pH set-up. This shows a maximal rate at 0 sec, which then decreases; this is the same trend for all set-ups. Finally, only at the 7C set-up did a plateau begin to appear, suggesting that this set-up results in the fastest consumption of the substrate.
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Conclusion The results of these experiments indicate that catalase is sensitive to reaction conditions, and that the operating ranges are rather narrow. Enzymes are proteins and are sensitive to changes in their shape, which are in turn more or less sensitive to the environment. Enzymes have in fact evolved to operate best under the living conditions of the organisms that produce them. To further illustrate this point, it would be interesting to carry out the same studies using catalase obtained from different organisms, especially those that live under extreme conditions.

References Goodsell DS. 2004."Catalase".Molecule of the Month. RCSB Protein Data Bank. http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb57_1.html. Retrieved 2 May 2011. Lamontagne B, Tremblay A, Elela SA. 2000. The N-terminal domain that distinguishes yeast from bacterial RNase III contains a dimerization signal required for efficient double-stranded RNA cleavage. Mol Cell Biol 20(4): 1104-1115. Matthew Attia, Providing Catalase Graphs, Gratis
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