Based on that of Bergmeyer in which the rate of decomposition of hydrogen peroxide by peroxidase, with guaiacol as hydrogen donor, is determined by measuring the rate of colour development spectrophotometrically at 436 nm and at 25^?C.

Unit Definition

That amount of enzyme which catalyses the conversion of one micromole of hydrogen peroxide per minute at 25^?C.

Reagents

A	0.1 M Potassium Phosphate Buffer pH 7.0 Dissolve 0.53g KH2PO4 [MM136.09] and 1.06g K2HPO4 [MM174.18] in distilled H2O, check pH to 7.0 and dilute to 100 ml. Store diluent on ice and equilibrate buffer at 25?C.
B	0.018 M Guaiacol Dissolve 22.3 mg guaiacol (MM124.14) in 10 ml distilled water. Store on ice and prepare fresh daily.
C	Substrate: Dilute 0.1 ml 30% hydrogen peroxide with distilled water to 120 ml and adjust A240 in 1 cm light path to 0.4 to 0.41 versus distilled H2O. Store the solution on ice and prepare fresh daily.
D	Enzyme Solution: Dissolve 5 mg enzyme / ml 0.1M ice cold potassium phosphate buffer pH 7.0 (refer reagent 3A above). Immediately before assay, dilute to yield approximately 0.2 units / ml ice cold buffer. (Approximately 0.040 to 0.045 DA 436 / minute.)

Procedure

Temperature = 25^?C
Wavelength = 436 nm
Light Path = 1 cm

Into a 1cm quartz cell, pipette the following:

Buffer	2.80 ml
Guaiacol	0.05 ml
Substrate	0.05 ml
Equilibrate at 25?C and monitor DA/minute
Enzyme at zero time	0.10 ml
Total reaction volume	3.00 ml

Record the rate of increase in absorbance at 436 nm using the linear portion of the curve after the initial lag phase.

Calculation

	DA 436 / min x 4 x Vt x dilution factor
Volume activity (U / ml) =
	e x Vs

Where

Vt = final volume of reaction mixture (ml) = 3.00
Vs = sample volume (ml) = 0.1
e = micromolar extinction co-efficient of tetraguaiacol (cm²/micro mol) = 25.5
4 = derived from unit definition & principle

Unit Conversion

The relationship between the ‘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

Reference

Bergmeyer H.U.: Methods of Enzymatic Analysis 1, Academic Press, New York. 2nd Edition (1974), page 495

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