Reaction Rates of Various Substrate Concentrations and Catalase Temperatures

Abstract

A catalyst is a substance that is used to increase the rate of chemical reactions without being completely used up or going through a chemical change itself. Catalase, an enzyme, acts as a catalyst. It’s found in high quantities in the liver, and accelerates the decomposition of hydrogen peroxide. During this reaction, water and oxygen gas is produced. Certain factors, such as temperature and amount of catalase, effect the rate of this reaction. This is likely the case because these sorts of elements will affect if an enzyme will bind to active sites.

In this set of experiments, the rate of which oxygen was produced when both the temperature and concentration of catalase varied was recorded. Our hypothesis for experiment one was that a higher concentration of substrate H2O2 (0.8%) would produce the most oxygen.

Our hypothesis for experiment two was that the warm catalase (~37 ˚C) would produce the most oxygen verses our other various temperatures, such as cold or boiled.

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The results supported these hypothesizes, showing that the average reaction time was about 34.73 ± 15.98 mL/sec for 0.8% concentration of substrate. The warm catalase was shown to have produced 10 mL of oxygen in 47.62 ± 15.06 sec on average. The results for experiment one show that a higher concentration of the enzyme accelerates the reaction, and produces oxygen much faster than the lower quantities. For experiment two, the results show that warm catalase produces more gas, unlike the boiled catalase that shows the reaction stops once catalase becomes denatured.

Introduction

Catalase is a very common enzyme found in many life forms such as animals, plants, and bacteria.

This enzyme is found in the heme group, which lets hemoglobin and myoglobin to bind oxygen with the iron atom (Strother & Ackerman, 1960). Catalase is important in cellular antioxidant defense, because one of its main functions is to breakdown hydrogen peroxide. This stops hydroxyl radical from forming (Ho, Ye-Shihetal., 2004). This is a positive process because hydroxyl radical is a very reactive oxygen species that damages organic molecules (Dey, Saptarshietal., 2019).

The rate in which catalase breaks down hydrogen peroxide is based on a few environmental factors, like shaking and substrate concentration, as well as innate factors of the reaction itself (Dawson etal., 1943). The focus of these experiments is to demonstrate the effects of physical factors, heat, and substrate concentration in a catalase and substrate reaction. Our hypothesis for the first experiment was that a higher concentration (0.8%) of substrate H2O2 solution would produce the most oxygen within two minutes. The null hypothesis being that there will be no change depending on the concentration.

Our hypothesis for the second experiment was that the warm catalase (~37 ˚C) would produce the most oxygen over the other temperatures, such as the cold (~0˚C), room temperature (~25˚C), or boiled (100˚C) catalases. Again, the null hypothesis for this experiment would be that there are no notable changes. These factors, such as heat to a certain extent, effect enzyme activity and will determine how fast the reaction rate will be.

Materials and Methods

For experiment one, a 600 ml beaker almost completely filled with water placed inside a bowl was used. In tandem with the beaker, a 10 mL graduated cylinder also filled with water was placed upside down and inside of the beaker. The 10 mL graduated cylinder acts as the chamber for the oxygen produced from the reaction. The shorter end of a U-shaped tube was then placed inside the beaker, where the tip is inserted into the graduated cylinder. A 50 mL Erlenmeyer flask is sat next to the beaker, and filled with 10 mL of substrate solution (0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2) and 10 mL of catalase/buffer solution. Once this is done, the rubber stopper from the U-shaped tube is placed in the flask. The mixture is then swirled until the entirety of the chamber (graduated cylinder) is filled with oxygen, or when two minutes has passed. Results are recorded after each concentration has run through a trail, the flask is then cleaned, and the next concentration is used.

For experiment two, the same procedure is repeated, only this time the only substrate concentration used is 0.8%. The boiled catalase (denatured, ~100˚C) is added to the flask. The flask is then swirled until the chamber (the graduated cylinder) is filled with oxygen or until two minutes has passed, then the results are recorded. The flask is cleaned, and this is repeated for the cold (~0˚C), room temperature (~25˚C), and warm (~37˚C) catalases. All graphs, tables, and a t-test used for p-values were made using Excel.

Results

Figure 1 and table 1 represents the results of experiment one; table 1 being the data and figure 1 being a graph of this data. As seen in figure 1 and table 1, on average 0.0% H2O2 substrate concentration produced 0.00 O2 mL/min ± 0, while the 0.1% substrate produced 2.10 O2 mL/min ± 0.88. For the 0.2% concentration, an average of 2.86 O2 mL/min ± 0.81 was recorded. For 0.4% H2O2, 13.78 O2 mL/min ± 8.66 was produced. Lastly, the 0.8% substrate released 34.73 O2 mL/min ± 15.98 on average.

As for figure 2 and table 2, these show the results of the second experiment. Firstly, the cold catalase (~0˚C) the average reaction rate was 34.70 O2 mL/min ± 24.59. For the room temperature catalase (~25˚C) the average rate of reaction was 33.31 O2 mL/min ± 16.63. Next, for the warm catalase (~37˚C) the reaction rate was 47.62 O2 mL/min ± 15.06. Finally, the boiled catalase (~100˚C) had a reaction rate of 0.05 O2 mL/min ± 0.14.

Table 3 shows the p-values of the second experiment. A t-test was used to find these values. This set of values is the relationship between the room temperature catalase and the other catalase temperatures. There is a high marginal significance between the room temperature verses warm temperature (p= 0.0930), and the room temperature verses cold temperature (p= 0.8965). Inversely, there is a low marginal significance for the room temperature verses boiled temperature (p= 0.0008).

Effect of change in substrate concentration on reaction rate Substrate concentration

Effect of change in temperature on reaction rate in a catalase mediated breakdown of H2O2

Temperature factor

Table 1 – Shows the average amount of oxygen (O2) produced from the reaction of room temperature catalase and various substrate concentrations.

Table 2 – Shows the average amount of oxygen (O2) produced from the reactions of varied temperatures of catalase and 0.8% substrate concentration.

Comparisons for p values temperature comparison used for t-test statistical analysis

p values

• p value for room temp vs warm temperature 0.093026299
• p value room temp vs cold 0.896507395
• p value room temp vs boiled 0.000768146

Table 3 - Analysis of statistical significance for each temperature condition vs room temperature (t-test: two sample assuming equal variances)

Discussion and Conclusion

For experiment one, the results show that the average reaction time for 0.8% concentration of substrate came up to be about 34.73 O2 mL/min ± 15.98, displaying the fact that the more concentrated the solution, the higher the rate of reaction would be. The standard deviation for this result is very high, showing that the values collected across the class were in a very big range. Despite that, this allows for the rejection of the null hypothesis. This is supported by past studies, on the fact that the addition of more catalase would raise the reaction rate of the solution (Dawson etal., 1943).

For experiment two, the warm catalase was shown to produce 10 mL of oxygen in 47.62 O2 mL/min ± 15.06 on average. The standard deviation for this result is also high. Again, due to the inaccurate results from the different groups. The p-values show a high marginal significance twice in the data. Once between the room temperature verses warm temperature analysis (p= 0.0930) and again between the room temperature verses cold temperature analysis (p= 0.8965). Since both values are not ≤ 0.05, this does not allow for the rejection of the null hypothesis for the second experiment. This is likely due to the errors in the recorded data. The numbers are skewed, and not entirely accurate. To make the results for the lab more precise for the future, flasks should be properly cleaned and the procedure should be followed more closely.

References

1. Dawson, Charles R. etal. 'ON THE INACTIVATION OF ASCORBIC ACID OXIDASE*'. Annals Of The New York Academy Of Sciences, vol 92, no. 1, 1961, pp. 212-222. Wiley, doi:10.1111/j.1749-6632.1961.tb46121.x.
2. Strother, G.K., and E. Ackerman. 'Physical Factors Influencing Catalase Rate Constants'. BiochimicaEtBiophysicaActa, vol 47, no. 2, 1961, pp. 317-326. ElsevierBV, doi:10.1016/0006-3002(61)90292-x.
3. Ho, Ye-Shihetal. 'Mice Lacking Catalase Develop Normally But Show Differential Sensitivity To Oxidant Tissue Injury'. Journal Of Biological Chemistry, vol 279, no. 31, 2004, pp. 32804-32812. American Society For Biochemistry & Molecular Biology (ASBMB), doi:10.1074/jbc.m404800200.
4. Dey, Saptarshietal. 'Pharmaceuticals And Personal Care Products: Waste Management And Treatment Technology'. 2019. Elsevier, doi:10.1016/c2017-0-03544-9.