Recombinant Human Myeloperoxidase Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after reconstitution.

• Assay Buffer: 50 mM NaH₂PO₄, pH 7.0
• Recombinant Human Myeloperoxidase/MPO (rhMPO) (Catalog # 3174-MP)
• Hydrogen Peroxide Solution, 30% (v/v) (H₂O₂) (Sigma, Catalog # H1009)
• Guaiacol (Acros Organics, Catalog # AC120192500)
• Clear StripWell Microplate (Costar, Catalog # 92592)
• Plate Reader with Absorbance reading capability (Model: Spectramax Plus by Molecular Devices) or equivalent

1. Prepare 100 mM guaiacol in Assay Buffer by shaking or stirring for 30 minutes at room temperature prior to use. Note: Protect guaiacol solution from light.
2. Dilute rhMPO to 1 µg/mL in Assay Buffer.
3. Dilute hydrogen peroxide from 30% to 0.00667% in Assay Buffer.
4. Load in a clear microplate 20 µL of 1 µg/mL of rhMPO and 30 µL 0.00667% hydrogen peroxide, and start the reaction by adding 50 µL of 100 mM guaiacol.
5. Read at 470 nm in kinetic mode for 5 minutes.
6. Calculate specific activity:

*Adjusted for Substrate Blank
**Derived using known concentrations of hydrogen peroxide ranging from 20 to 300 µM. Each point contains 10 µg/mL of rhMPO (an amount so that the reaction will be completed in a short period of time) and 50 mM guaiacol. After each reaction is complete, the product is measured at 470 nm (read endpoint about every 20 seconds to find the maximum absorbance for each point). The maximum values of Abs470 (y-axis) and pmol of hydrogen peroxide (x axis) for each point is plotted linearly (y = mx + b) and the slope is calculated (m). The conversion factor is derived from the following equation as a unit of pmol/OD. It is multiplied by 2 because one mol of hydrogen peroxide is equal to two mol of oxidized guaicol (product).

Conversion Factor = (1/slope(m)) x 2 = pmol/OD

• rhMPO: 0.020 μg
• Hydrogen Peroxide: 0.002%
• Guaiacol: 50 mM