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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

 

Materials

     

References

  1. Chance, B., and Maehly, A.C., Methods in Enzymology, 2, 773-775 (1955).
  2. Shannon, L.M. et al., Journal of Biological Chemistry, 241, 2166-2172 (1966).

 

07/13-1

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