Methodology for Enzymes

Polyphenol Oxidase
(Monophenol, dihydroxyphenylalanine: Oxygen oxidoreductase EC

Phenol oxidases are copper proteins of wide occurrence in nature which catalyze the aerobic oxidation of certain phenolic substrates to quinones which are autoxidized to dark brown pigments generally known as melanins. These enzymes are assumed to be single enzymes with broad specificity although there is some evidence for the presence of more than one phenol oxidase in certain tissues. Each individual enzyme tends to catalyze the oxidation of one particular phenol or phenolic compound more readily than others. The polyphenol oxidase (PPO)comprises of catechol oxidase and laccase. The activities of these enzymes are important with regard to (a) plant defense mechanism against pests and diseases and (b) appearance palatability and use of plant products. Fresh fruits, vegetables, mushroom etc. contain these enzymes considerably.

The intensely yellow 2-nitro-5-thiobenzoic acid (TNB) with an absorption maximum at 412nm reacts with the quinones generated through enzymatic oxidation of 4-methylcatechol (catechol oxidase) and 1,4 dihydroxybenzene (laccase) to yield colorless adducts. The decrease in the absorbance of yellow-color due to enzyme activity is measured.

» Citrate-phosphate buffer 0.2M (pH 6.0)
» Preparation of 2-nitro-5-thiobenzoic acid anion (TNB): Add 30mg sodium borohydride to a suspension of Ellman's reagent, i.e. 5,5-dithiobis (2-nitrobenzoic acid) (19mg) in 10mL water. Within 1h, the disulphide is quantitatively reduced to the intensely yellow, water-soluble thiol.  This solution is stable for at least one week when stored at 4°C.
» Preparation of the Quinine Solutions
   Dissolve 4-methyl-1,2-benzoquinone in double-distilled water in a 50mL volumetric flask by bubbling nitrogen gas until the quinone is completely dissolved.
    Prepare p-benzoquinone solution also in a similar manner. Both solutions are stable for 30min, a time sufficient to carry out the spectrophotometric assay.
» Substrate Solution
   4-methylcatechol (2mM) for catechol oxidase assay Quinol (1,4 dihydroxybenzene, 2mM) for laccase assay.
» Enzyme Extract
   Prepare first acetone powder of fresh plant tissue (see under indole acetic acid oxidase)
   To get a crude enzyme preparation, mix 100mg acetone powder with 2.5mL of 0.2M citrate phosphate buffer (pH 6.0), 1mL of 1% Triton X-100, 6.5mL of water and 500mg polyamide. Shake for 1h and filter. Use the filtrate as enzyme source.

Pipette out into clean 1cm cuvette of 1.4mL citrate 0.1M phosphate buffer (pH 6.0), 0.5mL of TNB and 1mL of the substrate solution.
The reaction is initiated by the addition of 0.1mL of enzyme preparation and immediately note down the absorbance at 412nm in a spectrophotometer already set.
Follow the decrease in absorbance at 30sec intervals and record.

Read the change in absorbance per minute from the linear part of the curve.
Calculate the enzyme units according to the following equation:
Units in the test = K X (ΔA/min)
where, K is 0.272 for catechol oxidase and 0.242 for laccase.
One unit of either catechol oxidase or laccase is defined as the enzyme which transforms 1mmol of dihydricphenol to 1mmol of quinone per min under the assay conditions. One unit is equivalent to the consumption of 1mmol of TNB.

The method described is rapid and sensitive than many other methods available.
The enzyme concentration required to get satisfactory linearity of time vs absorbance decrease has to be standardized.
The relation between rate and enzyme concentration is linear for all catechol oxidases; however, with laccase it is observed only for low concentrations.
A simpler method of assaying polyphenol oxidase is given below.
Add 2.5mL of 0.1M phosphate buffer pH 6.5, 0.3mL of catechol solution (0.01M) into cuvette and set the spectrophotometer at 495nm. Now add 0.2mL of enzyme extract and start recording the change in absorbance for every 30 seconds up to 5 min.
For the above procedure, the enzyme extract may be prepared by grinding 5g leaves with a mortar and pestle in about 20mL medium containing 50mM Tris-HCl, pH 7.2, 0.4M Sorbitol and 10mM NaCl. Centrifuge the homogenate at 20,000g for 10min. and use the supernatant for assay.

1.   Esterbaner, H, Schwarel, E and Hayn, M (1977) Anal Biochem 77 486.