The aim of this experiment is to:
Determine how different concentrations of substrate affect the enzyme activity of the enzyme catalase
Construct and interpret graphs of the data collected.
Many animal and plant tissues undergo catalase reactions. The various biochemical reactions yields hydrogen peroxide which is broken down by catalase into oxygen and water. The following is a typical equation governing catalase reaction (Roberts and King, 1987).
H2O2 2H2O + O2
Hydrogen peroxide Water Oxygen
In this experiment, a filter paper that has been coated with enzyme is placed into a beaker containing hydrogen peroxide; the substrate. The enzyme breaks hydrogen peroxide into water and oxygen. Oxygen gas will be observed to collect at the base of the beaker before eventually rising to the surface and in the process forming bubbles. The rate of enzyme activity is measured by the time it takes the filter to rise to the surface.
The rate of enzyme activity shall be determined using the following relationship:
Rate of enzyme activity =Distance (depth of hydrogen peroxide in mm)Time (in secs)
Factors that affect the rate of enzyme activity has been studied. According to Seager and Slabaugh (2014), factors that affect the rate of enzyme activity are enzyme concentration, substrate concentration, temperature, and pH level. When the concentration of the enzyme is increased relative to the concentration of the substrate, the rate of enzyme activity increases. However, when the concentration of the substrate is increased relative to the enzyme, the rate of enzyme activity increases but it levels off at some point. But when pH level is very low, the rate of enzyme activity is very low. Again, when the pH level is very high, the rate of enzyme activity is also very low.. Therefore, there is an optimum point at which the rate of enzyme activity is very high, that is when pH is neutral. It has also been found that the rate of enzyme reaction also increases with increase in temperature until it reaches a point when the rate of reaction levels off and start to decrease. There is an optimum temperature at which rate of enzyme reaction is very high (Panesar, Marwaha, and Chopra, 2010)
Note: Given the fact that hydrogen peroxide is a very corrosive substance, safety glasses were worn during the process of pouring it. Care was also taken to ensure there was no contact with the body.
The following materials were required in the experiment
Potato extract which was prepared before class. A 30g of potato was blended in 6omls of iced water, filtered and stored in ice
Hydrogen peroxide in concentrations of (0.1%, 0.3%. 0.5%, 1%, 2%, 3%)
Filter paper circles ( Whatman No.1 filter 2.5cm in diameter was used)
Note: The potato extract (catalase) was kept in an ice bath throughout the experiment
The concentration of hydrogen peroxide was recorded as shown in Table 1.
Three beakers, each with 0% (water) were set up as detailed in steps 3 to 8. The beakers were then set on one side. Next, the beakers were frequently checked to see if there were any changes in the position of the disk.
25ml of the 0.1% hydrogen peroxide was poured into a 30ml beaker, then the depth was measured and recorded as shown in Table 1.
Forceps were used to obtain one paper circle which was then immersed in the potato juice for 5 seconds
Excess juice was drained on the paper circle by touching on a paper towel for 10 seconds
Using forceps, the paper was then placed at the bottom of the beaker containing hydrogen peroxide. The oxygen produced got trapped in the fibers of the paper circle and thus causing it to rise to the surface.
A stop clock was used to measure the time taken by the circle to reach the surface. Timing was began as soon as the paper touches the hydrogen peroxide.
Time was recorded as shown in Table 1.
Steps 2-8 were repeated, twice using new hydrogen peroxide and a new paper circle for each trial.
The average time was calculated and recorded in Table 1
Steps 2-10 was again repeated this time using different concentrations of hydrogen peroxide (0.3%, 0.5%, 1%, 2%, and 3%)
The rate of activity was calculated for each concentration (mm per second) and the results were completed as shown Table 1.
The following equation was used to calculate the rate of activity
Rate of enzyme activity =Distance (depth of hydrogen peroxide in mm)Mean time (in secs)
Discussion and Conclusion
The results are just indicate of the enzyme activity in different concentration of substrates. However, there might have been errors in pH of solution, timing, errors about reading depth of H2O2, and the treatment of the paper circle. The experiment was about effect of substrate concentration on enzyme activity. Future work may involve studying the effect of temperature, pH, and enzyme concentration on the enzyme activity.
AlevelNotes.com. 2015. Factors affecting Enzyme Activity. [Online] Available at: http://alevelnotes.com/factors-affecting-enzyme-activity/146 [accessed 27 April 2015]
Panesar, P.S. Marwaha, S.S. and Chopra, H.K. 2010. Enzymes in Food Processing: Fundamentals and Potential Applications. New Delhi: International Publishing House.
Roberts and King. 1987. Biology: A Functional Approach. Students' Manual. Cheltenham: Thomas Nelson and Sons.
Saini, B.L. 2010. Introduction to Biotechnology. New Delhi: University Science Press.
Seager, S. and Slabaugh, M. 2014.Chemistry for Today: General, Organic, and Biochemistry. Belmont, CA: BROOKS/COLE CENGAGE Learning