Peroxidase UV Vis assay

The focus of our research is the activity of sea urchin ovoperoxidase. We are starting with Horse Radish Peroxidase (HRP) to develop a standard protocol for measuring the activity of a peroxidase.

Standard Mixing Procedure

The mixture of Peroxidase UV-Vis assays requires the use of a Guaiacol stock solution and a hydrogen peroxide stock solution.

The stock solutions are prepared as follows:

Guaiacol Stock Solution

49.5 microliters of Guaiacol is added to a 25 mL pH 8.0 buffer solution, which is comprised of 25 mL of DI water and a 8.0 pH buffer tablet that has been mixed thoroughly.

Hydrogen Peroxide Stock Solution

17 microliters of hydrogen peroxide is added to 1 mL of DI water.

Assay

5.0 mL Guaiacol stock solution is added to a vial.

10 microliters of the hydrogen peroxide stock solution is added to the Guaiacol stock solution.

The desired amount of peroxidase is then added to the assay before the UV-Vis is ran.

References

Purification and Assay publication