User:Michael F. Nagle/Notebook/Chem 571/2012/10/02

Objective

• Prepare reagents for horseradish peroxidase (HRP) assay
  • Complete HRP assay with and without gold nanoparticles to see if they interact with HRP's active site

Procedure

1. HRP procedure¹ was followed. Time was plotted against absorbance on the UV/Vis spectroscoper.
2. 1mg horseradish peroxidase was dissolved in 1mL distilled water.
3. A .2M buffer of sodium phosphate was made at a pH of 7.
4. .0017M hydrogen peroxide solution was made by adding 1mL 30% hydrogen peroxide to 100mL distilled water. 1mL of hydrogen peroxide solution was diluted to 50mL with 49mL of sodium phosphate buffer for 34μM solution.
5. 698μM iodophenol solution was made in 1mL DMSO, since it's not soluble in water. 2.5mM 4-aminoantipyrine (AAP) solution was prepared with the sodium phosphate buffer.
6. From the stocks, solution was prepared with 18mM iodophenol, 7.285μM AAP, 1.7mM H₂O₂ and 115nM HRP.
   1. Such a solution was analyzed for the 1st, 3rd and 4th runs.
   2. For the 2nd run, HRP was increased to 2.3μM while AAP was kept at 7.285μM
   3. HRP was increased to 50μM and AAP to 156.25 μM for the final trial.

Data

Discussion

1. Most of the day was spent trying to determine concentrations of HRP and AAP that would allow us to see the reaction over the course of 300 seconds. For the final trial, we used the same ones as the other lab group. Tomorrow we will continue with these concentrations, and add AuNPs. This will help us determine whether AuNPs deactivate HRP by interacting with it's active site.

References

1. Horseradish Peroxidase Assay. Gold Biotechnology Image:3760-Protocol HRP AbsorbanceAssay.pdf