Environmental Variables and Their Effect on Horseradish Peroxidase

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  • Topic: PH, Horseradish, Enzyme
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  • Published : April 22, 2012
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Environmental Variables and their effect on Horseradish Peroxidase

Abstract

There are several factors that can affect the rate of reaction of peroxidase such as temperature, pH, concentration of peroxidase present and whether or not it has been boiled. Our experimental data demonstrated that peroxidase activity peaked between 23 degrees Celsius and 32 degrees Celsius. We found the pH to be 7 for optimal activity. As far as the concentration is concerned our results showed that as the concentration of horseradish peroxidase doubled so did the rate of reaction. Lastly, the boiled horseradish peroxidase proved to be denatured and did not yield any activity while the normal extract proceeded to react in an upwards fashion. In conclusion, the rate of reaction of horseradish peroxidase was optimal at neutral, room temperature conditions and it increased with concentration.

Introduction

The presence of enzymes in our bodies controls the multiple events happening simultaneously by lowering the activation energy of chemical reactions. (1) These enzymes perform best under a certain set of environmental conditions. The purpose of this laboratory investigation is to examine the activity or horseradish peroxidase, an enzyme, under a wide range of circumstances and key in on the prime set of conditions of its ideal functionality. The hypothesis proposed is that activity of peroxidase will be at its best under standard rather than extreme settings. In this experiment we will test the hypothesis and narrow in on a result.

Materials & Methods

The Environmental Variables and their effect on Horseradish Peroxidase experiment is conducted in several parts. The first is figure out if concentration plays a part in the reaction rate. To begin this trial, we gathered 3 cuvettes in which we added horseradish peroxidase in the following amounts; .5 mL, 1.0 mL and 2.0 mL. In each of these cuvettes, we added 2.0 mL of H2O2, 1.0 mL of Guaiacol and enough pH 5 Buffer to fill each cuvette to 8.0 mL with all of the materials combined. Immediately after adding the materials in each cuvette, we placed it in the spectrophotometer to get an absorbance reading @ 500 nm in 20 second intervals commencing at 20 seconds and concluding at 120 seconds.

The next part of the experiment is to evaluate if temperature contributes or hinders the reaction rate of horseradish peroxidase. We gathered 8 cuvettes and added, 2.0 ml of H2O2 and 1.0 mL of Guaiacol to each of the first 4 and 4.0 mL of pH 5 Buffer and 1.0 mL of peroxidase to each of the next four. After, we placed one from each set into an ice bucket reading 4 degrees C and water bath reading 32 degrees C, and 48 degrees C. We left the last set at room temperature (23 degrees C). We allowed the four sets to remain for 20 minutes at their designated temperatures. After 20 minutes, we mixed each set of matching temperature cuvettes together, shook them quickly and immediately placed in the spectrophotometer, set to 500 nm, to retrieve readings for 20, 40, 60, 80, 100 and 120 seconds. The trials were done one set at a time since the reaction begins straightaway after mixing each set.

The third experiment was to analyze if pH had an effect on peroxidase activity. In four separate cuvettes, we added 2.0 mL of H2O2, 1.0 mL of Peroxidase, 1.0 mL of Guaiacol. Next, 4.0 mL of different buffer solutions were added to each cuvette (pH 3, pH 5, pH 7 and finally pH 9). Immediately after combining the materials, an absorbance reading was taken by a spectrophotometer at 20, 40, 60, 80, 100 and 120 seconds. The trials were done one set at a time since the reaction begins straightaway after mixing each set.

Last of all, a test on boiled versus normal peroxidase was conducted to examine whether boiling has an effect on reaction rate. Two tests were conducted; the first contained 4.0 mL of pH 5 buffer, 2.0 mL of H2O2, 1.0 mL of Guaiacol and 1.0 mL of normal peroxidase. The...
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