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.
|
Peroxidase |
|
| 4 guaiacol + 4H202 |
 |
Tetraguaiacol + 8H20 |
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
| Weight activity (U/mg material) = |
U/ml
|
|
mg enzyme / ml original solution |
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
Specialists in the Isolation and Purification of Horseradish
Peroxidase and a Select Range of Enzymes