Hydrogen Peroxide Decomposition by Peroxidase

As a potent by-product of metabolism, hydrogen peroxide (H2O2) molecules should be enzymatically decomposed quickly so that no damage is done to the cells. Peroxidases, enzymes belonging to class catalases, catalyzes the chemically break –down of H2O2 into its water and oxygen molecules. The chemical reaction is represented as:

2H2O2 (l) + enzyme → 2H2O (l) + O2 (g)                

            Theoretically, if the amount of oxygen gas molecules (P) evolved during the reaction can be measured, determining the activity of peroxidases is feasible. Although H2O2 decomposes spontaneously, the rate is accelerated by peroxidase which lowers the energy barrier in the reaction (Campbell and Reece, 2005). As per collision theory (Max, 1916), when the collisions between the reactant molecules are assisted by an enzyme, less energy is required for the chemical reaction to occur and collisions between the substrate molecules are more frequent, thereby increasing the reaction rates. Inferring from this theory, this lab hypothesizes that collisions will be more frequent between the substrate molecules when the latter’s concentration rises, thereby increasing the rate of the reaction. Since the enzyme concentration is the limiting factor, this lab further supposes that no free enzyme will be available due to active sites saturation at a particular substrate concentration, and further raising the substrate concentration will not affect the enzyme activity. Both the hypotheses are based on the Michaelis- Menten’s model for enzyme kinetics (Michaelis and Menten, 1913). The model explains the enzyme kinetics in the monomolecular/ first-order biochemical reactions like decomposition of H2O2

into water and oxygen. Therefore, this experiment aims to validate how the change in concentration of the substrate, H2O2, affects the rate of the reaction and the activity of the peroxidase.

Results

Table1: Numbers of bubbles of oxygen evolved in 1 minute when potato discs are added to

hydrogen peroxide solutions of different concentration.

Molar concentration of H2O2

0.5 mol dm-3

1.0 mol dm-3

1.5 mol dm-3

2.0 mol dm-3

Relative rate of reactions (bubbles/min)

1st

count

16

48

99

114

2nd

count

23

48

89

116

3rd

count

30

55

94

81

Average

23

50

94

104

Figure.1 The graph was obtained by plotting average reaction rates against each substrate concentrations. The plot illustrates the effect change in substrate concentrate has on the enzyme activity. The maximum reaction rate is represented as Vmax.

Discussion

            It is evident from the above plot (figure.1) that the rate of the reaction increases with the increase in a concentration of the substrate but their correlation is not linear. In this experiment, the independent variable was the substrate concentration while the dependent variable was the reaction rate. Peroxidase catalytically decomposes H2O2 into water and oxygen molecules. The proportionality can be explained as the rise in frequency of random collisions between active sites of enzyme and substrate molecules when the latter’s concentration is high. However, this increase is limited and change in the rate of reaction progressively declines because more enzyme-substrate complexes are formed due to free enzymes continually locked with substrate molecules when the concentration of H2O2

increases as the reaction progresses. This explains the graph obtained by plotting rate of reaction versus substrate concentration. Thus, the experimental data (Table.1) and the plot (figure.1) confirm the hypothesis made in the introduction section. The graph is similar to saturation curve given by Michaelis-Menten for an enzyme reaction. At a given point of time and particular substrate concentration (S), active sites of all the enzymes in the reaction solution will be saturated with substrate completely. Therefore, any rise in concentration will not affect the reaction rate. At this point, reaction attains maximum velocity (Vmax) that is mathematically given by the Michaelis-Menten equation: V1=  =, where V1 is the reaction rate at any time and KM is the Michaelis constant for the enzyme, peroxidase.

Errors

1. Manual errors should be considered since sizes of the potato slices could vary, thereby H2O2 concentration might differ among individuals resulting in discrepancies in readings.

2. The potato slices may not be of uniform sizes which explain any difference in substrate concentration. This could vary the reaction rate and readings of each experiment.

3. H2O2 is very reactive and some has already decomposed as soon as it formed into its constituents: water and oxygen in the sample. Henceforth, this might change the volume of H2O2 in the sample, the factor that will influence the results of the experiments and readings.

Conclusion

            The relationship between hydrogen peroxide concentration and peroxidase activity was established and was in accord with the Michaelis- Menten’s model of enzyme kinetics. In short, the enzyme activity increased with the rise in substrate concentrations until saturation point reached. Post-saturation, any rise in substrate concentration had no apparent effect on the enzyme activity. Experiments were carried out with caution to avoid contamination.

References

Campbell, N.A. and Reece, J.B., 2005. Biology. 7th. Ed Pearson Benjamin Cummings. Cape Town, pp. 172-180.

Menten, L. and Michaelis, M.I., 1913. Die kinetik der invertinwirkung. Biochem Z, 49, pp.333-369.

Trautz, M., 1916. Das Gesetz der Reaktionsgeschwindigkeit und der Gleichgewichte in Gasen. Bestätigung der Additivität von Cv‐3/2R. Neue Bestimmung der Integrationskonstanten und der Moleküldurchmesser. Zeitschrift für anorganische und allgemeine Chemie, 96(1), pp.1-28.