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In this article we will discuss about the tests for estimation of polyphenol oxidase enzyme activity.
Principle:
The intensely yellow 2-nitro-5-thio-benzoic acid (TNB) with an absorption maximum at 412 nm reacts with quinones generated through enzymatic oxidation of 4-methyl-catechol (catechol oxidase) and 1, 4 di-hydroxy-benzene (laccase) to yield colourless adducts. The decrease in the absorbance of yellow- colour due to enzyme activity is measured.
Materials:
(i) Citrate-phosphate buffer 0.2 M (pH 6.0)
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(ii) Preparation of 2-nitro-5-thio-benzoic acid anion (TNB):
Add 30 mg sodium borohydride to a suspension of Ellman’s reagent, i.e. 5, 5-dithiobis (2-nitro-benzoic acid) (19 mg) in 10 ml water. Within 1 h, 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.
(iii) Preparation of the Quinine Solutions:
Dissolve 4-methyl-1, 2-benzoquinone in double-distilled water in a 50 ml volumetric flask by bubbling nitrogen gas until the quinone is completely dissolved.
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Prepare p-benzoquinone solution also in a similar manner. Both solutions are stable for 30 min, a time sufficient to carry out the spectrophotometric assay.
(iv) Substrate Solution:
4-methyl-catechol (2 mM) for catechol oxidase assay Quinol (1, 4 di-hydroxy-benzene, 2 mM) for laccase assay.
(v) Enzyme Extract:
Prepare first acetone powder of fresh plant tissue (see under indole acetic acid oxidase) To get a crude enzyme preparation, mix 100 mg acetone powder with 2.5 ml of 0.2 M citrate phosphate buffer (pH 6.0), 1 ml of 1% Triton X-100, 6.5 ml of water and 500 mg polyamide. Shake for 1 h and filter. Use the filtrate as enzyme source.
Test Procedure:
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1. 1.4 ml citrate 0.1 M phosphate buffer (pH 6.0), 0.5 ml of TNB and 1 ml of the substrate solution is pipetted out into a clean 1 cm cuvette.
2. The reaction is initiated by the addition of 0.1 ml of enzyme preparation and immediately note down the absorbance at 412 nm in a spectrophotometer already set.
3. The decrease in absorbance at 30 sec. intervals is followed and recorded.
Calculation:
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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 × (∆A/min)
where, K is 0.272 for catechol oxidase and 0.242 for laccase.
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One unit of either catechol oxidase or laccase is defined as the enzyme which transforms 1 µmol of di-hydro-phenol to 1 µmol of quinone per min under the assay conditions. One unit is equivalent to the consumption of 1 µmol of TNB.
Notes:
1. The method described is rapid and sensitive than many other methods available.
2. The enzyme concentration required to get satisfactory linearity of time vs. absorbance decrease has to be standardized.
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3. The relation between rate and enzyme concentration is linear for all catechol oxidases; however, with laccase, it is observed only for low concentrations.
4. A simple method of assaying polyphenol oxidase is given:
Add 2.5 ml of 0.1 M phosphate buffer pH 6.5, 0.3 ml of catechol solution (0.01 M) into cuvette and set the spectrophotometer at 495 nm. Now add 0.2 ml 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 5 g leaves with a mortar and pestle in about 20 ml medium containing 50 mM Tris-HCl, pH 7.2,0.4 M Sorbitol and 10 mM NaCl. Centrifuge the homogenate at 20,000 g for 10 min and use the supernatant for assay.
