The Effect of Temperature on the Activity of Catalase

The purpose of the study was to test the effects of the availability of an enzyme, the pH, and temperature on the rate of chemical reaction.

Hypothesis

1. As the enzyme concentration increases, the rate of reaction increases

2. Increase in temperature increases the activity of an enzyme

3. Enzymes function optimally at specific temperatures

The background

An enzyme is a substance, mostly proteins, that catalyzes a chemical reaction without being consumed in the reaction. In other words, the enzymes are not used in the chemical reaction but they play a role in lowering the activation energy of the reaction (Doble & Gummadi, 2007).  Without enzymes, most cellular reactions would require additional energy, such as heat, to occur. Therefore, enzymes plays a critical role by allowing chemical reactions at the normal temperature of the cell. Substrates are molecules that the enzyme acts. For catalysis reaction to occur, the substrate has to bind to the active site of the enzyme to form a complex in a lock-and-key as well as hand-and-glove mechanisms. In the complex, the enzyme arranges the reacting molecules in a precise manner to allow for chemical changes to take place (Hemker, 2012). The final substance that is released from the enzyme-substrate complex is called a product. The dissociation of an enzyme-substrate complex yields an enzyme and the product. The product is of lower free energy and its configuration is different from that of reactants (Sinha, 2004). Therefore, it is released as it cannot compactly fit in the active sites of the enzyme.

The chemical reaction proceeds as follows:

Substrate(s) + enzyme                              end product (s) + enzyme

Where K1, K-1 and K2 are the reaction rates for each of the reactions

In a catalase reaction, the toxic hydrogen peroxide (H2O2) is converted into water (H2O) and oxygen (O2) which are harmless.

H2O2

+ catalase                                  H2O + O2 + catalase

By monitoring the production of oxygen, the catalase reaction can be measured. Many dyes react with active oxygen. In most cases, a colorless dye is used and a color change will be a measure of the presence of oxygen. The test is referred to a dye-coupled reaction. For instance, if the dye to be used is guaiacol, the color change from colorless to brown will indicate the presence of oxygen. In presence of oxygen, the dye is oxidized to generate an oxidized guaiacol that is brown.

Some of the environmental factors that affect the activity of an enzyme are temperature and pH. Since enzymes are proteins in nature, very high temperatures (above 40 degrees Celsius for animal enzymes) destroys the shape and activity of the enzyme (denaturation). Besides, extreme pH also denatures proteins. In very low temperatures, the enzymes are inactivated.

Materials and methods

The experiment was performed using the methods that is published in the Biology 110 Biology I molecular and Cells Laboratory manual, 6th edition, by K. Dalton.

Results

Effect of catalase availability

The first part of the experiment aimed at determining the effect of catalase availability in the rate of reaction. With addition of the increase in enzyme concentration, the rate of formation of the product increases. The increase in the formation of the product is indicated by the increase in the absorbance.

Table 1: The effect of catalase availability

Time (seconds)

Tubes 2 and 3

0.5 ml enzyme

Tubes 4 and 5

1.0 ml enzyme

Tubes 6 and 7

2.0 ml enzyme

20

0.11

0.182

0.384

40

0.146

0.346

0.692

60

0.227

0.484

0.845

80

0.300

0.578

0.925

100

0.363

0.636

0.965

120

0.418

0.670

0.985

Graph 1: Line of best-fit for the effect of availability of enzyme

The activity value of the each of the enzyme concentration is determined.

Enzyme activity = change in absorbance/ change in time

However, the values of enzyme activity are obtained from the slope of the line of best-fit.

For 0.5 enzyme concentration, slope (m) the enzyme activity is 0.0032

For 1.0 enzyme concentration, the enzyme activity is 0.0049

For 2.0 enzyme concentration, the enzyme activity is 0.0056

As the amount of enzyme increased, the rate of reaction also increased. However, at higher temperatures of 100 degrees Celsius, the rate of reaction if constant as the enzyme will have been denatured. The reaction rates are indicated in table 2.

Table 2: effect of catalase availability on reaction rate

Enzyme amount

Reaction rate

0.5 ml

0.0032 ml/second

1.0 ml

0.0049 ml/second

2.0 ml

0.0056 ml/second

Effects of temperature on enzyme activity

As the temperature increased, the rate of reaction increased as shown by results in table 3.

Table 3: The effect of temperature on enzyme activity

Time (seconds)

Tube 2 and 3

0 – 4 0C

Tube 4 and 5

Room temp.

Tube 6 and 7

37 0C

Tube 8 and 9

48 0C

Tube 10 and 11

100 0C

20

0.154

0.120

0.341

0.113

1.50

40

0.310

0.295

0.614

0.261

1.52

60

0.432

0.422

0.746

0.384

1.52

80

0.526

0.490

0.800

0.458

1.52

100

0.596

0.530

0.830

0.494

1.54

120

0.646

0.552

0.855

0.520

1.54

Graph 2: The effect of temperature on enzyme activity

The pH effects of catalase

The rate of reaction is optimum at the pH of 5. Above or below that pH, the enzymatic activity of catalase is impaired. As shown in table 4.

Table 4: pH effects of catalase

Time (seconds)

Tubes 2 & 3

pH 3

Tubes 4 & 5

pH 5

Tubes 6 & 7

pH 7

Tubes 8 & 9

pH 9

20

0.004

0.161

0.236

0.008

40

0.003

0.412

0.438

0.010

60

0.007

0.624

0.610

0.011

80

0.007

0.796

0.752

0.011

100

0.008

0.945

0.870

0.012

120

0.000

1.080

0.965

0.011

Graph 3: pH effects of catalase

Discussion

The results confirm the hypothesis that was stated for the experiment. First, the findings confirm that presence of a catalyst, for this case catalase, increases the rate of chemical reaction. Also, the study confirms that environmental factors such as pH and temperature affect the activity of an enzyme. However, the study did not establish a proper trend on how the rate of reaction varies with temperature. For instance, the reaction rate at temperature between 0-4 0C was higher than that of room temperature and temperature at 48 0C. It is expected that the rate of reaction would be the lowest at the lowest temperature and it would increase systematically to the optimum temperature of 37 0C. At 100 0C, the enzymatic activity was negligible depicting that the enzyme had been denatured. Secondly, the findings confirms the hypothesis that enzymatic activity is optimum at particular pH. For this case, the optimum pH is 5. The method used when testing the role of temperature on enzymatic activity could have had flaws. First, the standard could not have set the absorbance to the right value. However, the experiment remains significant in enzymology. When studying enzymes, it would be important to bear in mind the effects of temperature and pH on the activity of various enzymes.

References

Dalton, K. (nd). Biology 110: Biology I molecular & cells laboratory manual. 6th edition.

Doble, M., & Gummadi, S. N. (2007). Biochemical engineering. PHI Learning Pvt. Ltd..

Hemker, H. C. (2012). Handbook of synthetic substrates. Springer Science & Business Media.

Sinha, R. K. (2004). Modern plant physiology. CRC Press.