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The Effects of Temperature and Chelating Agents on Catechol Oxidation
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Section 403

Jen Sallee 

Andrew Clapper

March 26, 2007

Introduction

An enzyme is a catalytic molecule that speeds up the rates of specific reactions by as much as several million times.  Enzymes have the ability to chemically recognize, bind, and change specific reactants.  Enzymes usually remain unchanged, so the can mediate the same reaction repeatedly.  Most enzymes are a kind of protein.  Activation energy is the minimum amount of internal energy that a molecule must have before a reaction begins.  Activation energy is also known as an energy barrier, as well as the amount of energy needed in order to align chemical groups, destabilize electric charges, and break molecular bonds.  Enzymes typically offer a stable microenvironment that is more favorable for a certain reaction than the surrounding environment would normally be. 

            One example of an enzyme is catecholase.  Catecholase enhances the reaction rate of catechol by reducing the activation energy required for catechol to oxidize.  Catechol is a compound that exists beneath the skin of many plants, including apples and potatoes.  When it is exposed to air, the oxygen in the atmosphere oxidizes it.  When catechol is oxidized, one of the products of the reaction is benzoquinone, which acts as an antiseptic for the plant.  The reaction that takes place with the help of catecholase can be expressed as catechol + 1/2O2 =====catecholase= benzoquinone + H2O. 

            We observe that when produce is stored in a cooler or freezer, it will often go much longer without changing color.  It is possible that this observation is a result of the fact that the cooler temperature is preventing the catechol in the produce from oxidizing as quickly as it would at room temperature.  Also, we observe that apples and potatoes turn a brownish color when cut open, which may indicate enzymatic activity.  We also see that ethylenediamine tetraacetic acid (EDTA) is often listed in food contents as a preservative.  Perhaps the magnesium and calcium that EDTA binds are the cofactors used by the enzymes of bacteria and fungi that can spoil food.  We also notice that benzoquinone reflects light of orange wavelengths and absorbs light of green wavelengths, which offers us a method of measuring enzymatic activity by measuring light absorbance.

            It is hypothesized that enzyme kept at 37 degrees Celsius will show the most absorbance and therefore the most enzymatic activity, because colder temperatures will slow down the enzyme reaction, and higher temperatures will denature the enzyme.  It is also hypothesized that calcium and magnesium are the cofactors necessary in the functioning of enzymes in bacteria and fungi that spoil food, because EDTA binds to those two molecules, and it is used as a food preservative.  

 

Method

13 spectrophotometer cuvettes were used to hold the different mixtures used in the experiment.  A spectrophotometer was used to measure the absorbance of various mixtures at a wavelength of 540 nanometers.  18 mL of catechol solution was used as one of the reactants in the reaction that we measured.  10 mL of enzyme solution was utilized to catalyze the reaction.  531 mL of distilled water was used to prepare the catechol solution and to act as an environment for the reaction in the cuvettes.  2 mL of EDTA and 2 mL of phenyl thiourea (PTU) were used as possible chelating agents.  5 labeled pipettes were used to measure the contents of each cuvette while keeping them separate.  At least 14 pieces of parafilm were used in order to keep the contents of the cuvettes inside while the cuvettes were inverted and shaken.  Kimwipes were used to wipe the cuvettes off just before they were measured for absorbance in order to attain an accurate reading.  A cuvette rack was used to hold the cuvettes while they were not being measured.  37 and 60 degree Celsius water baths were utilized to control the temperature of the environment outside the cuvettes.  One uncooked potato was used as the source of catechol for the procedure.  A potato peeler was used to peel the skin off of the potato, because the skin contained very little unoxidized catechol.  A refrigerated blender was used to create a liquid solution that contained the catechol, and it was kept cold before use to prevent possible acceleration of the enzyme reaction.  500 mL of the cold distilled water was used to cause the cells and organelles in the potato to burst in order to free up the most catechol.  Keeping the water cold prior to the procedure prevented the acceleration of the enzyme reaction during the preparation phase.  Several squares of cheese cloth were used to strain the solid material from the potato mixture in order to improve handling. 

A potato weighing 174.2 g was weighed while unpeeled to prevent the enzyme reaction from beginning during the preparation phase.  The potato was peeled with a potato peeler because the skin had very little unoxidized catechol.  It was then cut into small pieces to facilitate blending.  500mL of cold distilled water was added to the potato pieces to cause the organelles in the potato cells to burst and yield the maximum amount of catechol.  The blender was then closed, and the contents were blended in 10 second bursts to prevent the contents from heating.  The contents were then strained with cheese cloth so that the resulting solution was more thin and easier to use the pipettes on.  Next, a container was filled to overflowing and closed to keep oxygen out and prevent the catechol in the solution from oxidizing.

The spectrophotometer was set to 540 nanometers and allowed to warm up for 15 minutes before being used.  9 cuvettes were labeled with numbers 1 through 9.  Cuvettes 1, 3, 5, and 7 received 1mL of enzyme solution, and 4mL of dH2O.  Cuvettes 2, 4, 6, and 8 received 2mL of catechol, 1mL of enzyme, and 2mL of dH2O.  Cuvette 9 received 5mL of dH2O.  The cuvettes always received the enzyme solution last in order to prevent the reaction from accelerating until we were ready to begin measuring the reaction. 

After the contents of each cuvette were placed in the cuvettes, cuvette 9 was sealed with parafilm.  It was then inverted once to mix the contents, and the exterior of the cuvette was wiped with a kimwipe.  The cuvette was then placed into the spectrophotometer, and the vertical white line on the cuvette was lined up with the mark on the sample compartment of the spectrophotometer.  The lid of the sample compartment was closed, and the zero knob was moved until the spectrophotometer read 0.0 absorbency.  Next, the initial absorbance of all the other cuvettes was measured after sealing, inverting, and wiping each tube. 

After the initial readings of all of the cuvettes, cuvettes 1 and 2 were placed into an ice bath.  A thermometer was used to constantly measure the temperature of each environment.  Cuvettes 3 and 4 were left on the lab bench at room temperature, which was 22.5 degrees Celsius.  Cuvettes 5 and 6 were placed in the 37 degree water bath, and cuvettes 7 and 8 were placed in a 60 degree water bath.  The cuvettes were shaken frequently during the experiment to ensure that the contents were properly mixed.  After ten minutes, the cuvettes were removed from their environments.  Each cuvette was then covered and inverted.  Next, the cuvettes were wiped with a kimwipe.  The parafilm was removed from the cuvettes, and cuvette 9 was used to calibrate the spectrophotometer.  The absorbency of each of the other cuvettes was then recorded. 

To conduct the cofactors procedure, first the cuvettes were prepared.  Cuvettes 1, 2, and 3 received all contents other than catechol, at first.  Cuvette 1 was to receive 1mL of enzyme, 2mL of catechol, and 2mL of EDTA.  Cuvette 2 received 1mL enzyme, 2mL catechol, and 2mL of PTU.  Cuvette 3 received 1mL of enzyme, 2mL of catechol, and 2mL of dH2O.  Cuvette 4 received 5mL of dH2O.  The enzyme solution and chelating agent were allowed to sit for a minimum of 10 minutes before the catechol was added, in order to ensure thorough mixing.  The cuvettes were inverted and shaken every 2 minutes during this period to further promote mixing.  After at least ten minutes of mixing, the catechol solution was added and initial measurements were taken following the same procedure as in the temperature procedure.  Next, the cuvettes were placed into a 37 degree water bath and ten minutes elapsed before a second set of data was recorded. 

Two data charts were created to record the data for the temperature and cofactor procedures.  The charts show the absorbance rate for each cuvette for the initial readings and the readings after ten minutes.  Taking two sets of readings for each procedure after a fixed elapsed time period allows us to compare the changes in absorbance between cuvettes.  The blank cuvettes, the cuvettes containing only water, in each procedure allow us to calibrate the spectrophotometer.  When the spectrophotometer is calibrated with the blanks, we are subtracting the amount of absorbance that occurs when only H2O is present in the cuvette.  This allows us to measure the amount of enzymatic activity indicated by absorbance while controlling for absorbance that is not due to enzymatic activity.   

 

Results

            In the temperature procedure, Cuvette 8 showed the largest increase in absorbance and therefore enzymatic activity (Graph 1, Table 1).   Cuvette 8 contained both catechol and the enzyme solution, and was placed in the 60 degree bath in between measurements.  Also, the trend in the data was that the higher temperature a cuvette was kept during the procedure, the more enzymatic activity it displayed through absorbance (Graph 1, Table 1).  Also, the cuvettes containing both catechol and the enzyme solution showed a greater increase in enzymatic activity as indicated by absorbance.  

In the cofactor procedure, the cuvette containing PTU showed the smallest amount of increase between measurements (Graph 2, Table 2).  The cuvette containing EDTA does appear to have inhibited the enzyme reaction, but not as much as the PTU (Graph 2, Table 2).   

 

Discussion

The hypothesis that the cuvette with both the catechol and enzyme solution that was placed in the 37 degree bath would result in the greatest amount of enzymatic activity was not supported, since the cuvette that contained both solutions and was placed in the 60 degree bath showed the greatest amount of enzymatic activity.  Also, the hypothesis that magnesium and calcium are the necessary cofactors for catecholase was not supported, because the cuvette that contained PTU inhibited enzymatic activity much more than EDTA.  

Although the hypothesis that the 37 degree bath would result in the most enzymatic activity was not supported, the fact that the cuvettes containing both catechol and the enzyme solution showed a greater increase in enzymatic activity as indicated by absorbance is consistent with the premise that the enzyme acts as a catalyst for the oxidation reaction.  We thought that the 60 degree bath would denature the enzyme in the enzyme solution, but it appears as though it did not. 

One possible area of weakness in the study is human error.  If the parafilm was left on some cuvettes for a different amount of time than others, the data may have been skewed because the reaction depends on the presence of oxygen.  Also, the accuracy of the measurement devices used in the procedure may have resulted in additional error. 

The absorbance of green light that was measured by the spectrophotometer indicated enzymatic activity, because the benzoquinone that results from catechol being oxidized reflects orange wavelengths and absorbs green. 

The blank cuvettes used in both procedures accounted for the green light that was absorbed by a cuvette with only water, and allowed us to calibrate the spectrophotometer so that only the green light that was absorbed as a result of enzymatic activity was measured. 

It appears as though the cofactor in catecholase is copper, because PTU binds to the copper in catecholase, and the data showed that the PTU prevented the catecholase from assisting the catechol in oxidation (Table 2, Graph 2).  

 

 

Appendix

Table 1: Temperature Procedure

 

 

 

 

Cuvette

Initial Absorbance

Absorbance After 10 Min.

Change

Enzyme Only and Ice Bath

0.56

0.6

0.04

Catechol and Ice Bath

0.76

1

0.24

Enzyme Only 22.5 Celsius

0.49

0.8

0.31

Catechol and 22.5 Celsius

0.63

0.98

0.35

Enzyme Only 37 Celsius

0.43

0.54

0.11

Catechol 37 Celsius

0.5

1.08

0.58

Enzyme Only 60 Celsius

0.34

0.42

0.08

Catechol 60 Celsius

0.57

1.18

0.61

Blank

0

0

0

 

Table 2: Cofactor Procedure

Cuvette

Initial Absorbance

After 10 Minutes

EDTA

0.8

1.21

PTU

0.44

0.5

H2O

0.74

1.37

Blank

0

0

 

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