SOP: Quantitative Estimation of Catalase in Vegetables

GIFSQ — Global Institute for Food Safety & Quality

Principle

Catalase decomposes hydrogen peroxide; the residual peroxide is measured iodometrically (the differences between the blank titre and the sample titres are the iodine solution equivalents of the H₂O₂ present at the respective times). Method A demonstrates the presence or absence of catalase; Method B estimates the catalase content accurately in terms of Kt, the first-order reaction constant (log base 10) determined at 0°C, divided by the grams of sample per 50 ml of reaction mixture. Pure catalase has a Kt of 40,000 to 60,000.

Apparatus required

• High speed blender
• Volumetric flask
• Cotton gauze
• Quick-delivery pipettes
• 125-ml conical flasks
• Stainless steel or horn spatula (for the sample)

Reagents — relatively stable

a. Phosphate buffer, 0.1 M, pH 6.95: dissolve 15.22 g of dibasic potassium phosphate (K₂HPO₄·3H₂O) and 4.54 g of monobasic potassium phosphate (KH₂PO₄) in water and dilute to 1 litre.

b. Sulphuric acid containing molybdate (H₂SO₄–molybdate solution): add 5.5 ml of H₂SO₄ to about 800 ml of water, cool, add 0.1 g of finely ground ammonium molybdate ((NH₄)₆Mo₇O₂₄·4H₂O), mix until completely dissolved, and dilute to 1 litre with water.

c. Sodium thiosulphate solution, approximately 0.01% in 10% potassium iodide: dissolve 100 g of potassium iodide, 2.5 g of sodium thiosulphate and approximately 1 g of sodium carbonate in 500 ml of water and dilute to 1 litre. This solution need not be standardised.

d. Iodine solution, 0.1 N: prepare as given in the standard-solutions chapter (Chapter 35 of the book). Avoid exposure to excessive light even when taken in a burette.

Reagents — relatively unstable

e. Hydrogen peroxide, 0.1 N (H₂O₂): dilute 0.58 ml of 30% H₂O₂ to 100 ml with cold water; store in a refrigerator or ice bath when not in use; prepare daily.

f. Glucose solution, 20% in phosphate buffer: dissolve 20 g of glucose in 100 ml of phosphate buffer solution; store in a refrigerator; stable for 1 week.

g. Starch indicator: dissolve 1 g of starch in 100 ml of cold water and heat to boiling; prepare once in 15 days.

Sample preparation

Blend 50 g of fresh or frozen vegetable in a high speed blender with 100 ml of water and 1 g of calcium carbonate for 3 min, transfer to a volumetric flask and make up to 200 ml. Strain through cotton gauze and assay the filtrate within 30 min (the print reads “3C min”; carried as 30 min, flagged). Use only a stainless steel or horn spatula for the sample.

Procedure

Method A: To demonstrate the presence or absence of catalase

Dilute 10 ml of the filtrate (or less, depending on activity) with water to 43 ml

↓

Add 5 ml of glucose buffer solution and mix

↓

Add 2 ml of 0.1 N H₂O₂, mix thoroughly, and quickly pipette 10 ml of the reaction mixture with a quick-delivery pipette into a 125-ml conical flask containing 10 ml of H₂SO₄–molybdate solution; this is the zero-time aliquot (zero-time is the time when the delivery of the aliquot from the pipette is started)

↓

Remove 10 ml aliquots at 5 and 10 min intervals in the same way

↓

Keep the temperature of the reaction mixture at less than 20°C throughout (catalase activity is about twice as great at 20°C as at 0°C, but non-enzymatic decomposition of H₂O₂ is also greater; above 20°C, catalase is destroyed by H₂O₂ in addition to non-enzymatic decomposition)

↓

Within an hour, add 5 ml of Na₂S₂O₃–KI solution to each flask and mix

↓

After 3–5 min, titrate the excess sodium thiosulphate with 0.01 N iodine, using about 10 drops of starch indicator

↓

Run a blank in the same way with water added in place of H₂O₂ (no 5 and 10 min aliquots are needed for the blank)

Method B: To estimate the catalase content accurately in terms of Kt

Carry out the assay exactly as in Method A, but maintain the reaction mixture at 0°C; in addition to the 0, 5 and 10 min values, determine the titre value at the end of 15 min also.

To determine the activity of an enzyme preparation rather than a vegetable, dissolve a suitable amount of the preparation in dilute buffer of pH 6.5–7.5, preferably containing dextrose.

Calculation

The gap between the blank titre and each sample titre tells how much H₂O₂ was still present at that time, expressed as its iodine-solution equivalent.

• The zero-time titre value should be between 0.5 ml and 2 ml of iodine solution.
• The difference between the blank titre value and the zero-time titre value should be 3.0 to 4.5 ml of 0.01 N iodine. When the difference is less than 3.0 ml (iodine titre values greater than 2 ml), it indicates that the H₂O₂ was too weak, or that the catalase activity was so high that a large amount of H₂O₂ was decomposed before the zero-time aliquot was removed (in the latter case the 5 and 10 min differences may be nearly 0 although large amounts of catalase are present); in such a case, use Method B.
• Using 10 ml aliquots of extract, when the blank titre value is 4–6 ml, the zero-time titre value is less than 2.0 ml, and the 5 and 10 min values do not differ from the zero-time titre value by more than 0.2 and 0.4 ml respectively, the catalase may be considered absent within the experimental error.

Kt is calculated using the following expression:

Kt = [(2.303 / (t₂ − t₁)) × log₁₀[(blank titre − titre at t₁) / (blank titre − titre at t₂)]] / (g of sample per 50 ml of reaction mixture)

• Where,
• t₁ and t₂ = the initial and final times for the 2 titre values under consideration
• blank titre = the titre value of the aliquot containing no H₂O₂
• g of sample per 50 ml of reaction mixture = ml of extract used × 0.25 (50 g of sample are made to 200 ml in the sample preparation, so 1 ml of extract = 0.25 g of sample; the print reads “0.25 ml”, carried as 0.25, flagged)

Note: the printed layout of the Kt formula is garbled in the available copy; the expression above is read from the book’s own definitions (first-order constant, log base 10, blank titre, and grams of sample per 50 ml of reaction mixture), flagged for checking against the printed book.

• The value of Kt for the 0 to 5 min period and for the 5 to 10 min period should check at 0°C; at higher temperatures Kt for 5 to 10 min may be lower than Kt for 0 to 5 min, as catalase is inactivated by H₂O₂.
• For accurate results, the enzyme concentration should be such that the difference between the blank titre and the zero-time (t₀) titre is 3 to 4 ml, and the difference between the blank titre and the t₁₀ titre is 0.5–1.0 ml of iodine solution.

Precautions

• Keep the reaction mixture below 20°C in Method A and at 0°C in Method B.
• Within an hour of removing each aliquot, add the Na₂S₂O₃–KI solution to its flask (then titrate 3–5 min after adding).
• Avoid exposing the iodine solution to excessive light, even in the burette.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition. Cobey, H.S. jr., and Manning, G.R., Quick Frozen Foods, 15 (10), 54 (1953).