HPA Project

Project Overview

For this project, the goal was to quantify the concentration of HPA remaining in solution after a reaction between hydrogen peroxide, horseradish peroxidase, and HPA is completed. To do this we made standards of known concentrations of HPA, made reaction vials of the hydrogen peroxide, enzyme (horseradish peroxidase), and HPA. We ran these through the HPLC to collect the data.

My research partner, Will Fox, ran a similar experiment with acetaminophen, the link to his page can be found here.

HPA General Information

IUPAC name: 2-(4-Hydroxyphenyl)acetic acid

HPA is a model substrate for oxidative processes. It is also involved in the metabolic processes of some plants. HPA was chosen for this experiment because it is biologically active, has a phenol group that is easily oxidized, and the peroxidase doesn't contribute to the effects of this chemical.

Physical Properties of HPA

Molecular weight: 152.149

Melting point: 150°C

Boiling point: 346.6°C

Density: 1.3 g/mL

pKa (predicted): 4.5

Described as a beige powder.

Making the Samples

This project required HPA standards to be made. The following link describes this process.

HPA Standards

Our actual samples to be tested required the making of a 2 mM HPA pH 5 buffer stock solution, a hydrogen peroxide stock solution, and a horseradish peroxidase solution.

2 mM HPA Buffer Stock Solution

Put the 2 mM HPA standard into a bottle and add a pH 5 buffer tablet. Stir until dissolved.

Hydrogen Peroxide Stock Solution

Measure 5 mL of water into a scintillation vial. Add 283 microliters of 30% hydrogen peroxide to the water. This provides a hydrogen peroxide solution of about 0.413 M.

Horseradish Peroxidase Stock Solution

Preparing the Final Samples

Measure out 5 mL of the 2 mM HPA buffer stock solution into 4 different scintillation vials. Add hydrogen peroxide solution in different amounts to each vial (3 microliters, 6 microliters, 12 microliters, and 24 microliters). Add 10 microliters of horseradish peroxidase solution to each vial.

Instrumentation

After making the standards, we tested the standards using the HPLC to see where the peaks were for the different concentrations of HPA.

The following link goes into more depth about working with the HPLC:

HPLC

What the HPLC Tells Us

We ran these samples through the HPLC at the wavelength of 275 nm, which is the lambda max for HPA. Running the samples at this wavelength tells us how much the samples absorb at this wavelength.

Data Analysis

To analyze the HPLC data, the data was exported as an arw file. This file was then loaded into Igor, which we used to make chromatograms. Chromatograms allowed us to gain a better understanding of the how each concentration of HPA absorbs at 275 nm for our standards, and how each sample absorbs at 275 nm. We used the information from the chromatograms to make standard curves in excel. The slopes of the standard curves gave us an equation we could use to find the concentration of a sample using the absorbance.

Example standard curve data can be found here.

Equation

The following equation was used to find the concentration of HPA in our samples. The equation is solved for x, which is concentration, then the absorbance is plugged in for y and solved.

y = 0.0486x + 0.0021

R² = 1

Chromatogram Data

Figure 1. Graph of the samples tested with hydrogen peroxide in different concentrations catalyzed by horseradish peroxidase.

IGOR Data Files

This is the IGOR file for the simulation of the Gepasi Data.

File:gepasi_sim.pxp

This is the IGOR file for the graph of the Gepasi data which gave us absorbances.

File:geprates.pxp

This is the IGOR file for the graph of the differing concentrations of hydrogen peroxide in the HPA/HRP reaction.

File:allsamplesupdated.pxp

Gepasi

We used the software Gepasi to analyze the kinetics of the reaction between horseradish peroxidase (HRP) and hydrogen peroxide. Gepasi simulates how the compounds will react with each other. The following link describes how to run Gepasi:

Gepasi

We used several different HRP reactions to collect data from Gepasi. These reactions can be found here.

Gepasi Graphs

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  Figure 2. Graph of the different HRP phases during the reaction.

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  Figure 3. Graph of the different HRP concentrations when the rate of the HRP2 was increased.

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  Figure 4. Graph of the different HRP 1 concentrations when the rate of the HRP2 was increased.

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  Figure 5. Graph of the different HRP 2 concentrations when the rate of the HRP2 was increased.

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  Figure 6. Graph of the different AH radical concentrations when the rate of the HRP2 was increased.

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  Figure 7. Graph of the different AH radical concentrations when the rate of the dimerization was increased. This graph has the conditions of HRP 2 rate at 1e4.

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  Figure 8. Graph of the different AH radical concentrations when the rate of the dimerization was increased. This graph has the conditions of HRP 2 rate at 1e5.

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  Figure 9. Graph of the oxidation products with differing rates.

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  Figure 10. Simulation of the oxdation product data from Gepasi.

Copasi

We used the software Copasi to analyze the kinetics of the reaction between horseradish peroxidase (HRP) and hydrogen peroxide. Copasi simulates how the compounds will react with each other. The following link describes how to run Copasi:

Copasi

Data Summary

From Gepasi we learned that the enzyme is recycled much more quickly when the rate of the HRP 2 is increased. This means only a small amount of the enzyme is required when the rate of the HRP 2 is higher.

ChemSketch

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  Figure 11. The chemical structure of HPA.

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  Figure 12. The chemical structure of one of the oxidation products "AA."

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  Figure 13. The chemical structure of one of the oxidation products "BB."

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  Figure 14. The chemical structure of one of the oxidation products "AB."

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  Figure 15. The chemical process of the radical resonance.

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  Figure 16. The chemical structure of one of the oxidation products "triAH2."

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  Figure 17. The chemical structure of one of the oxidation products "triisoAH2."

References

HPA Oxidation Reactions

Immobilized Enzyme Article