Materials and Reagents

Aluminum foil
Hydrogen peroxide, 35% in water (H₂O₂) (TCI, catalog number: H1222, CAS RN: 7722-84-1)
L(+)-ascorbic acid (CAS 50-81-7) (Merck Millipore, catalog number:100468)
0.1 M potassium phosphate buffer (pH 7.0) (see Recipes)
Reaction mixture (see Recipes)

Equipment

Scale
Pipettes
Stirrer
Mortar and pestle
Centrifuge machine
Double beam UV-VIS spectrophotometer with Graphic LCD – Typr 2205
Glassware: test tubes, beakers, conical flask, measuring cylinder, and a volumetric flask (Slisco scientific and Ace Glass Incorporated)

Procedure

Sample extraction
Note: The complete extraction procedure must be carried out at 0–4 °C. The ascorbate peroxidase activity was measured in leaf tissues of sorghum plants.
1. Completely homogenize 2 g of fresh and cleaned tissue sample in 10 mL of fresh 0.1 M potassium phosphate buffer (pH 7.0) by using a previously chilled mortar and pestle.
2. Centrifuge the homogenate at 11,180 × g for 15 min in a temperature-controlled centrifuge machine.
3. Collect the supernatant as crude extract and discard the pellet completely.
4. Use this enzyme extract immediately for enzyme assay and preserve the remaining extract in the refrigerator for total soluble protein estimation, which is required for enzyme specific activity calculation.
Enzyme assay
Note: APX is assayed by the method of Nakano and Asada (1981). For estimating APX activity use freshly prepared reagents and extracts only.
1. Add 3 mL of reaction mixture (see Recipes) and initiate the reaction by adding 50 µL of enzyme extract (from step A4) at the end.
2. The blank can be simultaneously prepared for each sample by taking 50 µL of boiled enzyme extract instead of enzyme extract.
3. Record the decrease in absorbance at 290 nm using the spectrophotometer for 2 min against a suitable blank.
4. The enzyme activity can be calculated using the molar extinction coefficient (absorbance of one molar solution) of 2.8 mM^-1 cm^-1 for ascorbate in the standard equation given below. Enzyme activity will be given in enzyme units, with one enzyme unit corresponding to the amount of enzyme required to oxidize 1 nmol of ascorbic acid per minute.
  
  Standard equation for absorbance: A = ϵ × l × с
  
  Where A is the amount of light absorbed by the sample at a given wavelength, ϵ is the molar extinction coefficient, l is the distance that the light travels through the solution, and с is the concentration of the absorbing species.
Note: To get better and more accurate results according to your samples and instruments, calibration between L-ascorbate, H₂O₂, and enzyme extract should be performed before starting the final procedure.

Calculations:
The enzyme activity and specific activity can be calculated as explained in Table 1 for a supposed sample size. The total protein content of the sample is required to calculate the specific activity, which can be determined by Lowry’s method.

Table 1. Ascorbate peroxidase activity (units or nmol of ascorbate/minute)

Supposed sample used: extract was made from a 2 g sample of fresh leaves in 10 mL of potassium phosphate buffer; from this, 0.05 mL of extract was used for assay. Assume P mg/mL of protein was found per sample for the present case from Lowry’s method.
Absorbance at 290 nm						Enzyme activity and Specific activity
Replicate	Time 0 s	15 s	30 s	45 s	60 s	Decrease in absorbance	Average decrease in absorbance	Enzyme activity/minute (units) = activity/0.05 mL	Dilution factor (activity/mL of extract)	Total protein content (mg/mL)	Specific activity (µmol/min/mg)
1st	a	b	c	d	e	a - e = F	(F + O + X) / 3 = Y	Y / 2.8 = Z	(Z / 0.05) × 1 = G	P	G / P = Q
2nd	j	k	l	m	n	j - n = O
3rd	s	t	u	v	w	s - w = X

Recipes

0.1 M potassium phosphate buffer (pH 7.0)
0.5 L of 1 M K₂HPO₄ at 174.18 g mol^-1 = 87.09 g
0.5 L of 1 M KH₂PO₄ at 136.09 g mol^-1 = 68.045 g
Mix 61.5 mL of 1 M K₂HPO₄ with 38.5 mL 1 M KH₂PO₄ for the preparation of 0.1 M potassium phosphate buffer pH 7.0 at 25 °C
Reaction mixture
2.7 mL of 100 mM potassium phosphate buffer (pH 7.0)
0.1 mL of L(+)-ascorbic acid
0.15 mL of H₂O₂

Acknowledgments

This research was financially supported by CCS Haryana Agricultural University authority. The corresponding author deeply appreciates the CCSHA University and its staff members for their valuable assistance in experimental conductance and data analysis. My gratitude also goes to Dr. H. R. Singal for encouraging me to write this research article.

Competing interests

The authors declare that they have no conflicts of interest.

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