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

Progress as of December 2018

The main focus of the first semester of this research project has been to develop an efficient lab protocol for measuring the activity of a peroxidase. Since we will eventually be working with sea urchin peroxidase, background research was done on how to properly care for sea urchins as well as how to make UV-Vis assays for sea urchin peroxidase. With this information, we developed a standard mixing protocol for the UV-Vis assay using HRP. Several weeks have been spent modifying this procedure to produce more consistent results. Brad has taught me how to use the kinetics function for the UV-Vis, how to analyze data using Igor, and how to use the mixing attachment for the UV-Vis. We have also set up the tank which will hold our sea urchins when they arrive next semester.