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HomeEnzyme Activity AssaysSpectrophotometric Determination of Reinheitszahl (RZ) for Peroxidase (EC 1.11.1.7)

Spectrophotometric Determination of Reinheitszahl (RZ) for Peroxidase (EC 1.11.1.7)

Description

Reinheitszahl (RZ) is the ratio of absorbance due to hemin (A403, Soret region) to absorbance due to protein (A275). This ratio (A403/A275) is not a measure of enzyme activity. Products using this method include, but are not limited to, P1709, P2088, P6140, P6782, P8125, P8170, P8250, P8375, P8415, and P8651.

Reagents and Equipment Required

1.0 M Potassium phosphate monobasic solution (Product No. P8709)

1.0 M Potassium phosphate dibasic solution (Product No. P8584)

Cuvettes and thermostatted spectrophotometer

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions
(Storage/Stability)

Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.

Phosphate Buffer (100 mM Potassium Phosphate Buffer, pH 6.0 at 20 °C) – Add 17.36 mL of 1.0 M Potassium phosphate monobasic solution (Product No. P8709). Add 2.64 mL of 1.0 M Potassium phosphate dibasic solution (Product No. P8584) and adjust to final volume of 200 mL using ultrapure water. Adjust to pH 6.0 at 20 °C using 1 N KOH or 1 N HCl. Store the Phosphate Buffer on ice.

Peroxidase Solution – Prepare a 10 mg/mL solution of peroxidase in COLD Phosphate Buffer.

Procedure

1. Pipette the following reagent into quartz cuvettes:

2. Record the initial absorbance at 403 nm and 275 nm using a suitable spectrophotometer.

3. Then add:

4. Mix by inversion and then record the final absorbance at 403 nm and 275 nm using a suitable spectrophotometer.

Results

Calculations

RZ Factor
Materials
Loading

References

1.
Chance B, Maehly A. 1955. [136] Assay of catalases and peroxidases.764-775. https://doi.org/10.1016/s0076-6879(55)02300-8
2.
Shannon L, Kay E, Lew J. 1966. Peroxidase isozymes from horseradish roots I. Isolation and physical properties.. Journal of Biological Chemistry. 241(9):2166-2172.
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