Finding the Optimal pH of the Enzyme Peroxidase with the Aid of a Spectrophotometer

Finding the Optimal pH of the Enzyme Peroxidase with the Aid of a Spectrophotometer

ABSTRACT

The peroxidase enzyme was partially purified, and the enzyme activity was calculated with the use of a spectrophotometer, demonstrating the effect of pH on peroxidase activity. The objective of this exercise was to determine the ideal pH for peroxidase activity. The data concluded that the optimum pH for peroxidase activity is 5, and the “standardized” enzyme volume is 2.8mL.

INTRODUCTION

The human body works hard to keep everything running and in tact. When substances enter the body that are foreign, the body fights to get rid of these toxins. For example, hydrogen peroxide is toxic to cells, so it is important for the cell to get rid of it. Enzymes are in control of converting reactions into products. The enzyme peroxidase works to destroy the hydrogen peroxide in the cell. It does this by converting the hydrogen peroxide into oxygen and water.

Peroxidase releases oxygen during the procedure. When the rate of how much oxygen is being produced is measured, we can measure the rate of the peroxidase enzyme. Guaiacol is a dye that turns brown when oxygen is supplied.

In this experiment, the tissue from a turnip will be homogenized and filtered to get most of the enzyme peroxidase. The peroxidase will mix with the hydrogen peroxide and the enzyme activity will be measured using the spectrometer. I think that with a higher pH will create a higher reaction rate and a lower pH will create a lower reaction rate due to the acidity.

MATERIALS AND METHODS

I followed the methods in the Cells and Molecules Lab Manual on pages 82-87.

RESULTS

Table 1: Reaction Mixtures for Standardization of Peroxidase; Absorbance Data
Tube
pH 5 buffer Peroxidase solution
Guaiacol solution
Turnip extract total volume
Absorbance
20” 40” 60” 80” 100” 120”
1
3.0 mL
1.0 mL
1.0 mL
0
5.0 mL
0.000
2
2.8 mL
1.0 mL
1.0 mL
0.2 mL
5.0 mL
.441 .716 .946 1.143 1.302 1.489
3
2.5 mL
1.0 mL
1.0 mL
0.5 mL
5.0 mL
.871 1.299 1.625 1.797 1.907 1.949
4
2.0 mL
1.0 mL
1.0 mL
1.0 mL
5.0 mL
1.265 1.747 1.932 1.994 2.057 2.092

Table 1 shows the absorbance of four test tubes at 20 second intervals, spanning a total of 2 minutes. The first tube was the control, and the remaining three tubes had different amounts of pH 5 buffer in each. They also had different amounts of turnip extract in each. All tubes, including the constant, contained 1.0mL of peroxide solution. All tubes also contained 1.0mL of guaiacol solution, bringing the total volume of each test tube to 5.0mL.

Graph 1

Figure One shows the rate of change of absorbance at 500 nm for each test tube over a span of two minutes. The constant had an absorbance of 0 over the entire two minute span, while test tubes two, three, and four demonstrated an increase in absorbance over every 20 second interval. The slopes of test tubes two, three, and four are approximately .99, .86, and .61 respectively.

Table 2: Reaction Mixtures for Measuring the Effect of pH on Peroxidase Reaction Rate; Absorbance Data

Tube pH buffer
Peroxide solution
Guaiacol solution
Turnip extract
Total volume
Absorbance
20” 40” 60” 80” 100” 120”
1
5
3.0 mL
1.0 mL
1.0 mL
0
5.0 mL
0.0000
2
3
2.8 mL
1.0 mL
1.0 mL
0.2 mL
5.0 mL
.311 .556 .754 .900 1.018 1.107
3
5
2.8 mL
1.0 mL
1.0 mL
0.2 mL
5.0 mL
.465 .876 1.154 1.338 1.530 1.663
4
7
2.8 mL
1.0 mL
1.0 mL
0.2 mL
5.0 mL
.396 .624 .808 .942 1.046 1.099
5
9
2.8 mL
1.0 mL
1.0 mL
0.2 mL
5.0 mL
.245 .378 .511 .612 . 688 .767

Table two shows the absorbance of five test tubes at 20 second intervals, spanning a total of 2 minutes. The first tube was the control, containing 3.0mL of pH buffer 5 and 0mL of turnip extract. The remaining tubes contained 2.8mL of each different pH buffer: 3, 5, 7, and 9. They contained 2.8mL of pH buffer, which was the “standardized” enzyme volume determined from the data in Table One.

Graph 2

Graph 2 shows the rates of absorbance for the buffers pH 3 and pH 5. The slope of pH 3 is approximately .73 and the slope for pH 5 is approximately .92.

Graph 3

Graph 3 shows the rate of absorbance for the buffers pH 7 and pH 9. The slope for the line of pH 7 is approximately .67 and the slope for the line of pH 9 is approximately .51.

Table 3: Slope of line of each reaction pH Slope
3
0.0073
5
0.0092
7
0.0067
9
0.00505

Table three shows the slope of the lines of each absorbance for each pH buffer.

DISCUSSION

In this laboratory experiment, enzyme activity was measured using a spectrometer. From my data, I concluded that the optimal pH for a high reaction rate is pH 5. My hypothesis turned out to be wrong because pH 5 had the best reaction rate. The higher the pH the less of a reaction rate there was. An issue that my group came across was that the spectrometer wasn’t functioning properly but that issue was resolved.