Effect of an Acidic Fluid on Enzymatic Activity

Acid Denaturation of Catalase

The enzyme catalase is an integral component of endogenous antioxidant defenses in both plants (Blokhina, Virolainen, and Fagerstedt, 2003) and animals (Hermes-Lima and Zeneno-Savin, 2002). These defenses are required to keep reactive oxygen species (ROS) in check, otherwise accumulation would result in harm to cells and tissue. ROS species include the superoxide radical (O2), hydrogen peroxide (H2O2), hydroxyl radical (HO), singlet oxygen, ozone, lipid peroxides, and nitric oxide. However, under conditions of oxidative stress, ROS species can accumulate and threaten cellular and tissue health. For example, hypoxia causes H2O2 to accumulate in the roots and leaves of some plants (reviewed by Blokhina, Virolainen, and Fagerstedt, 2003) and in mammalian cells, over 100 genes involved in antioxidant defense are induced (reviewed by Hermes-Lima and Zeneno-Savin, 2002).

Some enzymes are able to withstand extreme conditions, in terms of pH and temperatures. Although catalase activity has been studied extensively in the past, including under extreme conditions, we believe that assays for catalase activity need not depend on expensive laboratory equipment like ultracentrifuges and spectrophotometers (e.g., Samejima, Miyahara, Takeda, Hachimori, and Hirano, 1981) and can therefore be used for high school and college level laboratory instruction. As a proof of principle, the effect of acid on the ability of catalase to convert H2O2 to oxygen and water will be tested using relatively simply and safe methods. The hypothesis is that acidic conditions below pH 3.5 will stop catalase activity using this assay, a result consistent with previously published results (e.g., Goldblith and Proctor, 1950).

Materials and Methods

Catalase Activity Assay -- A number of reliable methods have been developed in the past to measure catalase activity, including the use of permanganate and spectrophotometers (e.g., Goldblith and Proctor, 1950), but today the concentration of H2O2 can be measured semi-quantitatively very quickly and simply using test strips (Macherey-Nagel, 2011). The Quanofix® Peroxide 1000 test strips can measure peroxide concentration up to 1000 ppm, which is 1/30 the concentration of commercially available 3% H2O2 solutions.

Materials List:

3% H2O2 from local pharmacy

Distilled water

15 ml glass test tubes pH-Fix test strips #92115, Macherey-Nagel

1.0 N. HCl sol. In water, w/dropper

Glass stir rods, scalpel, tweezers, measuring pipets

Quantofix® Peroxide 1000 test strips #91333, Macherey-Nagel

1.0 N. NaOH sol. In water, w / dropper

One piece of fresh beef liver, not previously frozen

Phosphate-buffer (11.9 mM Phosphate buffer, pH 7.0)

Room temperature water bath scale

Marking pens for glass tubes

To 6 test tubes labeled p1 through p6, add 2.9 ml of phosphate buffer and set aside at room temperature. To 6 test tubes labeled r1 through r6, aliquot 2 ml of the 3% H2O2 solution. To tubes r2 through r6, add increasing amounts of 1 N. HCl, starting with 1 drop for test tube r2 and ending with 5 drops in test tube r6. Carefully swirl the tubes to mix the solutions and place in the room temperature water bath. Check the pH of each tube with the pH test strips.

Cut the ice-cold beef liver into 6 equal sized slices (about 1 cm3) or chunks using the scalpel. Ensure the pieces are approximately equal in size by weighing them, then adjusting as necessary with the scalpel. Record the weights obtained. Drop a single piece of ice-cold beef liver into each reaction test tube (r1-r6), 3 minutes apart. Incubate in the water bath for exactly 20 minutes.

At the end of the incubation period, swirl the reaction tube and transfer 100 ul of the solution into a test tube containing 2.9 ml of phosphate buffer. Swirl to mix and measure the peroxide concentrations using the Quantofix® peroxide test strips. Each measurement must be taken and recorded within the 3-minute interval, so that the next sample can be diluted, measured, and recorded at the end of the 20 min. incubation. After the assay is completed, add 1N NaOH to tubes r2 through r6 to neutralize the solutions (1 drop to tube 2, ending with 5 drops added to tube 6) prior to discarding the contents of the tubes.

Results

The catalase assay was performed in triplicate and all data measurements are shown in the Table. A pH gradient was created by adding increasing amounts of HCl to the reaction tubes, as can be seen by the test strip readings. Liver piece weights were also fairly uniform, with averages across samples almost identical. The liver pieces were capable of converting most of the H2O2 to water and oxygen at near neutral pH, but at pH values less than 3.5, catalase activity is stopped. The effect of pH on catalase activity can be seen graphically in the Figure.

Table: Raw Data and Averages for Catalase Assay in Triplicate

pH Readings

Liver Weights

Peroxide Conc. (ppm)

Samples

1st

2nd

3rd

Avg

1st

2nd

3rd

Avg

1st

2nd

3rd

Avg

1

6.5

6.0

6.5

6.3

1.1g

1.0g

1.1g

1.1

2

4.5

4.5

4.5

4.5

1.1g

1.1g

1.2g

1.1

3

3.5

3.5

3.0

3.3

1.2g

1.0g

1.0g

1.1

4

3.0

2.5

3.0

2.8

1.0g

0.9g

1.1g

1.0

1,000

1,000

5

2.0

2.5

2.0

2.2

1.0g

1.0g

1.2g

1.1

1,000

1,000

1,000

1,000

6

2.0

1.5

2.0

1.8

1.2g

1.2g

1.1g

1.2

1,000

1,000

1,000

1,000

Figure: Catalase Activity by Acid pH. The x-axis represents the average pH levels obtained in the test tubes, while the y-axis represents average H2O2 levels in ppm. Error bars represent standard error.

Discussion

As shown in the Figure, under acidic conditions the catalase activity supplied by the beef liver pieces is lost. This result supports our hypothesis that acidic conditions stops catalase conversion of H2O2 at approximately the pH value defined previously (Goldblith and Proctor, 1950).

Based on the work of Samejima and colleagues (1981), catalase activity is lost in acidic conditions in part because tertiary structure is lost. However, they also showed that subunit dissociation is not sufficient for irreversible loss of catalase activity, since rapid neutralization of solutions above pH 3.0 allows most of the catalase activity to be reconstituted. Longer incubation times at low pH, or pH below 2.5, will irreversibly destroy catalase activity, theoretically by disrupting the association between ? -- helical structures and heme groups. The result presented in the Figure therefore probably represents two different mechanisms, one depending on disruption of tertiary structure, and the second on disruption of the secondary structure. This could be tested by first neutralizing the solutions and then adding more peroxide.