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 (Catalog Number P8709)

1.0 M Potassium phosphate dibasic solution (Catalog Number 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 (Catalog Number P8709). Add 2.64 mL of 1.0 M Potassium phosphate dibasic solution (Catalog Number 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:

Reagent	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
Phosphate Buffer	2.90	2.90	2.90

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

3. Then add:

Reagent	Test 1 (mL)	Test 1 (mL)	Test 1 (mL)
Peroxidase Solution	0.10	0.10	0.10

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

Results

Calculations

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.