Enzymatic Activity

Abstract:

The objective of this lab was to determine the effects of certain variables on enzymatic activity and to get a quantitative feel for the effectiveness of a certain enzyme, in this case peroxidase obtained from potatoes. In order to do this, we conducted experiments in which the effects of boiling and inhibitors were examined by comparing normal enzymes to those affected by the factors being tested. Using a spectrophotometer, we analyzed the absorbance levels of the enzymes at various time intervals to determine how the enzymatic activity changed over time, as the enzyme's catalytic effects began to be observable. Based on our results, we concluded that inhibitors and boiling both effectively disrupt enzymatic activity, reducing the normally linear reaction to nearly nonexistent.

Introduction:

Enzymes are proteins that act as biological catalysts. Because they speed up metabolic processes that would otherwise take too long to occur, they are fundamental to an organism's ability to function. Because their ability to function depends on the conditions of their environment, it is important to understand how they are affected by different variables.

Because hydrogen peroxide is often created as a by-product of activity in the peroxisomes, it is necessary for it to be detoxified. The enzyme that carries out this function is peroxidase, which converts toxic hydrogen peroxide to harmless water and oxygen. It is this enzyme that we studied in this lab, analyzing the effects of various factors.

In this experiment, we tested the effects of boiling and competitive inhibitors, and hypothesized that both will reduce enzymatic activity. Boiling should reduce activity because the high temperature should denature the enzyme, and the inhibitors should do so because the competition they provide reduces the ability of the enzyme to react with the molecules it is supposed to react with.

Methods:

In our analysis of enzyme activity, we studied the effects of inhibitors and boiling on peroxidase activity. Before conducting experiments to test these two factors, we first had to standard the amount of enzyme. The laboratory instructor prepared the extract, substituting potatoes for the turnips. She weighed out 2 g of potatoes without the skin, and cut them into small chunks. Next she homogenized the tissue by adding 300 mL of 4oC 0.1M phosphate buffer at pH7, and then blended the mixture in a cold blender. Finally, it was filtered through a cheesecloth into a flask covered in foil, and was kept on ice for the duration of the experiment.

After preparing the extract, the amount of the enzyme to be used for the other experiments had to be standardized. Four 50 mL beakers were filled with the following solutions, with a corresponding pipette for each one: pH 5 buffer, hydrogen peroxide, the extract, and guaiacol. Seven test tubes were prepared as follows:

Table 1: Mixing table for standardization

Test tube # pH 5 buffer (mL) H2O2 (mL) Extract (mL) Guaiacol (mL) Total volume (mL)

1 5.0 0 2.0 1.0 8

2 0 2.0 0 1.0 3

3 4.5 0 0.5 0 5

4 0 2.0 0 1.0 3

5 4.0 0 1.0 0 5

6 0 2.0 0 1.0 3

7 3.0 0 2.0 0 5

The contents of test tube 1 are used to blank the spectrophotometer to zero absorbance at 500 nm. The contents of tubes 2 and 3 are mixed together, and then poured into a cuvette. Six readings are taken at 20 second intervals to record the absorbance. The process is repeated for tubes 4 and 5, and again for tubes 6 and 7.

To test for the effects of competitive inhibitors, five drops of 1% hydroxylamine are to be added to the enzyme extract. Five tubes were prepared as follows:

Table 2: Mixing table for inhibitors

Test tube # pH 5 buffer (mL) H2O2 (mL) Extract (mL) Guaiacol (mL) Total volume (mL)

1 5.0 0 2.0 1.0 8

2 0 2.0 0 1.0 3

3 3.0 0 2.0 0 5

4 0 2.0 0 1.0 3

5 3.0 0 2.0 0 5

The five drops of hydroxylamine were added to test tube 5, and the mixture was allowed to sit for five minutes. Test tube 1 was used to blank the spectrophotometer, and then test tubes 2 and 3 were mixed together, poured into a cuvette, and measurements were taken every 20 seconds for two minutes. The process was repeated for test tubes 4 and 5.

Finally, the effects of boiling were tested by putting 4 mL of extract in a test tube and putting it in a boiling water bath. Three test tubes were prepared as follows:

Table 3: Mixing table for boiling

Test tube # pH 5 buffer (mL) H2O2 (mL) Extract (mL) Guaiacol (mL) Total volume (mL)

1 5.0 0 2.0 1.0 8

2 0 2.0 0 1.0 3

3 3.0 0 2.0 0 5

The contents of tube 1 were used to blank the spectrophotometer, and then the contents of tubes 2 and 3 were mixed, poured into a cuvette, and readings were taken for six intervals of 20 seconds each.

Results:

Standardization

Test tubes 2 and 3 had results which were negligible, and while the results for 4 and 5 were much better, both paled in comparison to the effectiveness of the mixture obtained from 6 and 7. Because of the superior absorbance when 2.0 mL of extract were used, this amount becomes the standard with which all extract amounts are based on.

Table 4: Results for standardization

Time

(sec)

Tubes 2 & 3

(0.5 mL extract)

Tubes 4 & 5

(1.0 mL extract)