Methodology for Enzymes

(Donor: H2O2 Oxidoreductase E.C.

Peroxidase (POD) includes in its widest sense a group of specific enzymes such as NAD-Peroxidase, NADP-Peroxidase, fatty acid peroxidase etc. as well as a group of very non-specific enzymes from different sources which are simply known as POD (donor: H2O2-oxidoreductase POD catalyses the dehydrogenation of a large number of organic compounds such as phenols, aromatic amines, hydroquinones etc. POD occurs in animals, higher plants and other organisms. The best studied is horse radish POD.

Guaiacol is used as substrate for the assay of peroxidase.
Guaiacol 4- H2O2
Oxidized guiacol + 2H2O

The resulting oxidized (dehydrogenated) guaiacol is probably more than one compound and depends on the reaction conditions. The rate of formation of guaiacol dehydrogenation product is a measure of the POD activity and can be assayed spectrophotometrically at 436nm.

» Phosphate Buffer 0.1M (pH 7.0)
» Guaiacol Solution 20mM
Dissolve 240mg guaiacol in water and make up to 100mL. It can be stored frozen for many months.
» Hydrogen peroxide solution (0.042% = 12.3mM). Dilute 0.14mL of 30% H2O2 to 100 mL with water. The extinction  of this solution should be 0.485 at 240nm. Prepare freshly.
» Enzyme Extract
    Extract 1g of fresh plant tissue in 3mL of 0.1M phosphate buffer pH 7 by grinding with a pre-cooled mortar and pestle. Centrifuge the homogenate at 18,000g at 5°C for 15 min. Use the supernatant as enzyme source within 2—4h. Store on ice till the assay is carried out.

Pipette out 3mL buffer solution, 0.05mL guaiacol solution, 0.1mL enzyme extract and 0.03mL hydrogen peroxide solution in a cuvette. (Bring the buffer solution to 25°C before assay).
Mix well. Place the cuvette in the spectrophotometer.
Wait until the absorbance has increased by 0.05. Start a stop-watch and note time required in minutes (Δt) to increase the absorbance by 0.1.

Since the extinction coefficient of guaiacol dehydrogenation product at 436nm under the conditions specified is 6.39 per micromole, the enzyme activity per liter of extract is calculated as below:

Enzyme activity units/liter =
3.18 x 0.1 x 1000
6.39 x 1 x Δt x 0.1


1.  Most accurate values are obtained when Δt is between 1 and 3min. The enzyme extract has therefore to be diluted appropriately.
2.  O-dianisidine (1mg/mL methanol) may be used as an alternative substrate for the assay. The oxidized O-dianisidine (yellow/orange colored compound) is measured at 430nm. Take 3.5mL phosphate buffer (pH 6.5) in a clean dry cuvette. Add 0.2mL enzymes extract and 0.1mL freshly prepared O-dianisidine solution. Bring the assay mixture to 28-30°C and then place the cuvette in the spectrophotometer set at 430nm. Then, add 0.2mL 0.2M H2O2 and mix. Immediately start the stop watch. Read the initial absorbance and then at every 30sec intervals up to 3min. If the rate of increase is very high, repeat the assay with diluted extracts. Plot increase in absorbance against time. From the linear phase, read the change in absorbance per min. Express enzyme activity in terms of rate of increased absorbance per unit time per mg protein or tissue weight. A water blank is used in the assay.

1. Putter, J (1974) In: Methods of Enzymatic Analysis 2 (Ed Bergmeyer) Academic Press New York p 685.
2. Malik, C P and Singh, M B (1980) In: Plant Enzymology and Histoenzymology. Kalyani Publishers New Delhi p 53.