Specialists in the Isolation and Purification of Horseradish Peroxidase and a Select Range of Enzymes

Assay Procedure (Ref. No. FGAP026) - Peroxidase (Purpurogallin Spectrophotometric Units)

The method of assay measures the oxidation of pyrogallol to purpurogallin by peroxidase when catalysed by peroxidase at 420 nm and at 20^°C.

	Peroxidase
Pyrogallol + H202		purpurogallin

Unit Definition

One unit of peroxidase is defined as the amount of enzyme required to catalyse the production of 1 mg of purpurogallin from pyrogallol in 20 seconds at 20^°C under the assay conditions described.

Reagents

1.	1M NaOH: Dissolve 20 g NaOH [MM40.0] in ± 450 ml distilled H2O. Cool solution and adjust volume to 500 ml with distilled H2O.
2.	0.1M Phosphate Buffer pH6.0: Dissolve 13.6g of KH2PO4 [MM 136.09] in ± 800 ml of distilled H2O. Adjust to pH 6.0 with 1M NaOH. Adjust volume to 1000 ml [in a volumetric flask]. This buffer solution is stable for up to one month at 2°C to 8°C.
3.	5.33% Pyrogallol solution (m/v): Dissolve 533 mg pyrogallol [MM 126.11] to a concentration of 53.3 mg/ml in H2O. Prepare a fresh solution daily.

Substrate [H₂O₂ solution]

Dilute 1 ml of 30% H₂O₂ to a final volume of  75 ml with distilled H₂O. Check A₂₄₀ by diluting 1:15. Reading should be ±0.4. Adjust if necessary. Prepare a fresh solution daily.

Enzyme Solution

Dissolve freeze dried peroxidase to a concentration of 10 mg / ml 0.1 M potassium phosphate buffer pH 6.0 [2]. Immediately prior to assay, dilute appropriately in buffer [2] to yield 0.5 - 1.5 u/ml [0.25 < DA_{420/20 secs} < 0.6].

Procedure

Temperature = 20^°C
Wavelength = 420 nm
Light path = 1 cm

Pipette the following volumes into a 10 mm cuvette:

0.1M pPhosphate buffer [2]	2.40 ml
Pyrogallol solution [3]	0.30 ml
H2O2 solution	0.20 ml
Equilibrate in water bath until temperature reaches 20°C
Add enzyme at zero time and mix	0.10 ml
Total reaction volume	3.00 ml
and start recording immediately

Immediately start recording the increase in absorbance at 420 nm. Measure the change in absorbance per 20 seconds over the linear portion of the curve and use this value in the calculation.

Calculation

	DA 420 / 20 sec x Vt x Df
Volume activity (U / ml) =
	e x Vs

Where

e = molar extinction coefficient  = 12.0
Vt = final volume of reaction mixture = 3.00 ml
Df = Dilution factor
Vs = sample volume = 0.10 ml
Ce = Enzyme concentration in mg/ml

Hence:
Volume activity (U/ml) = DA₄₂₀/ 20 sec x 2.5 x dilution factor

Weight activity (U/mg material) =	U/ml
	mg enzyme / ml original solution

Unit Conversion

The relationship between 'purpurogallin unit' and the 'guaiacol unit' has been reported to be approximately 1:1. In our laboratory we have determined that, one purpurogallin unit as described above multiplied by 0.909 is equivalent to the guaiacol unit as per procedure FGAP001. That is

Purpurogallin Unit x 0.909 = Guaiacol Unit or

Guaiacol Unit x 1.1 = Purpurogallin Unit