Peroxidase Enzyme Lab

Description: A peroxidase enzyme, which was extracted from a brassica compestris (turnip), is tested under various conditions in temperature, pH level, and competitive inhibitor (hydroxylamine).

ABSTRACT:

In order to determine the properties of an enzyme, a peroxidase enzyme was extracted from a brassica compestris (turnip) and tested under various temperatures, pH levels, and by a competitive inhibitor (hydroxylamine). The enzyme activity was measured in various ways depending on the activity. Temperature effects showed the amount of activity and determined the optimal pH level. The spectrometer showed the absorbance units at 500nm and determined the optimal temperature. The temperatures tested where 4ºC (approximately refrigerator temperature),
23ºC (approximately room temperature), 32ºC, and 48ºC. The pH levels tested were; pH3, pH5, pH7, and pH9. The competitive inhibitor used was Hydroxylamine. These tests resulted in the ability to determine the optimal temperature: 32ºC because it closest to body temperature. The best pH level was found to be pH5.

INTRODUCTION

An enzyme is a protein that serves as a catalyst, which is a chemical agent that changes the rate of a reaction without being consumed by the reaction.
The enzyme dramatically speeds up reactions by lowering the activation energy barrier. Every enzyme has a unique shape because the substrates that bond to it are all different shapes. It fits like a lock and key. Once the substrate enters the active site, the enzyme slightly changes shape in order to lock in the substrate and holds it in place. Enzymes allow organisms to live by increasing the rate of a chemical reaction. The peroxidase enzyme catalyzes the reaction of the formation of oxygen. Various temperatures and pH levels will be tested in order to determine the optimal environment for the peroxidase
enzyme.

HYPOTHESIS:

Temperature: If the optimal human body temperature is 36ºC, then the enzyme 's optimal temperature should be similar.

Boiling: If most proteins are denatured when they are heated to a temperature above 70ºC, then the enzyme will be dead when boiled.

pH: Because the organisms are extremely sensitive to changes in pH levels, the peroxidase enzyme too will be extremely sensitive.

Hydroxylamine: Because hydroxylamine has a similar shape to the substrate, hydrogen peroxide, it will bond to part of the enzyme and block the substrate from entering. This would be measured by decrease of absorbance units.

MATERIALS AND METHODS

Please see citations for details: (Dolphin, 2005)

*Changes to lab protocol:

page 81. Preparing an extract containing peroxidase; step 1. 1 gram instead of 10.

page 81. Instead of adding 100ml at once, add 30ml, grind and then add 70ml.

page 81. Standardizing the Amount of Enzyme: label 3 beakers instead of 4.

page 87. Instead of no control, there was a control of tube that had a pH5, 2.0 H2O2mL, 0mL extract, 0mL hydroxylamine treated extract, 1.0mL Guaiacol, and a total volume of 8mL.

RESULTS

Please refer to included tables and graphs.

The following are attached:

Table 1/Figure 1: Volume of Turnip Extract

Table 2/Figure 2: Temperature Graph

Table 3/Figure 3: Temperature Derivative Graph

Table 4/Figure 4: pH Graph

Table 5/Figure 5: pH Derivative Graph

Table 6/Figure 6: Boiled Extract

Table 7/Figure 7: Hydroxylamine Graph

Based on the graph (Figure 2), it was determined that the most optimal temperature for the peroxidase enzyme was 32ºC. It was also determined that the enzyme functions to its highest potential at the pH 5 level. That is shown in Figure 4. In addition, Figure 6 proved that the enzyme becomes denaturized when heated above 70C. Lastly, the enzyme inhibitor, hydroxylamine, resulted in a decrease of absorbance units.

DISCUSSION

The hypothesis that was originally stated regarding the various temperatures needs to be slightly modified.
The optimal temperature is not exactly the same to the human body however it is relatively close (four degrees difference).

The hypothesis for the effect of various pHs on the activity of the peroxidase enzyme was not clear enough and did not predict any results. Based on the experiment, the best pH for the peroxidase enzyme is pH 5.

Meanwhile, the hypothesis for the effect of boiling the extract was correct. The results obtained in the experiment supported the original hypothesis that when proteins (which an enzyme is) are heated to a temperature above 70ºC, the enzyme will be dead when it is boiled.

Based on the effects of the hydroxylamine treated extract, the original hypothesis is again accepted. Because the inhibitor, hydroxylamine, blocked the substrate of hydrogen peroxide from entering the active site, the absorbance units decreased compared to the normal extract absorbance units.

Further work that would be needed to test explanations would include a more variety of temperatures as well as other enzymes and compare the results between various enzymes.
CITATIONS

Dolphin, Warren D. 2005. Biological Investigations. Lab Topic 7 Determining the Properties of an Enzyme, ed. Kent A. Peterson, pp.79-89.

New York, NY: McGraw-Hill