Ascorbate peroxidase assay

Protocol

 

Authors

Askim Hediye Sekmen, Ismail Turkan

Overview

This protocol outlines measurement of APX activity in plant tissue by spectrophotomeric assay.

Background – Antioxidant enzymes

Plants, being aerobic organisms, utilize molecular O2as a terminal electron acceptor. As a reduction, highly reactive intermediates, reactive species (ROS), are produced. ROS such as singlet oxygen (O21), superoxide (O2-.) and hydrogen peroxide (H2O2) are normal products of metabolism and are produced in all cellular compartments within a variety of processes. In general, they are maintained at constant basal levels in healthy cells. However, they can destroy normal metabolism through oxidative damage of lipids, proteins, and nucleic acids when they are produced in excess as a result of oxidative stress (Gill and Tuteja, 2010). To overcome oxidative stress, together with non-enzymatic antioxidant molecules (ascorbate, glutathione, -tocopherol etc.), plants detoxify ROS by up-regulating antioxidative enzymes like superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; E.C 1.11.1.6), peroxidase (POX; EC1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) (Turkan and Demiral, 2009). SOD provide the first line of defense against the toxic effects of elevated levels of ROS. The SODs converts O2-.to H2O2. The hydrogen peroxide produced is then scavenged by catalase and a variety of peroxidases. Catalase dismutates H2O2into water and molecular oxygen, whereas POX decomposes H2O2by oxidation of co-substrates such as phenolic compounds and/or antioxidants. APX is involved in scavenging of H2O2in water-water and ASH-GSH cycles and utilizes ASH as the electron donor. GR is a potential enzyme of the ASH-GSH cycle and plays an essential role in defense system against ROS (Gill and Tuteja, 2010; Ahmad et al., 2010). This protocol is one of a number of ANTIOXIDANT ENZYME PROTOCOLS:

PROTOCOL: Superoxide dismutase assay
PROTOCOL: Catalase assay
PROTOCOL: Peroxidase assay
PROTOCOL: Glutathione reductase assay

Background – Ascorbate peroxidase

In this protocol, APX (EC 1.11.1.11) activity was measured according to Nakano and Asada (1981). The assay depends on the decrease in absorbance at 290 nm as ascorbate was oxidized.

Materials/Equipment

a) Chemical Materials

  • NaH2PO4& Na2HPO4
  • Ethylenediamine tetraacetic acid (EDTA)
  • Polyvinylpolypyrrolidone (PVPP)
  • Ascorbate (ASC)
  • H2O2
  • Liquid nitrogen

b) Apparatus and Equipments

  • pH meter
  • Mortar and pestle
  • Various micropipettes
  • Eppendorf tubes (1.5 ml)
  • Spectrophotometer
  • Centrifuge
  • Quartz cuvette

Procedure

a) Solutions

Extraction Buffer 50 mM Na-P Buffer (pH 7.0) 2 % PVPP 0.1 mM EDTA (MW: 292.2 g/mol) 2 mM Ascorbate (MW: 176.13 g/mol) Total Volume: 100 ml

  • 50 mM Na-P buffer (pH 7.0), 100 ml
  • 2% PVPP, 100 ml

    2 g PVPP in 100 ml extraction buffer

  • 0.1 mM EDTA, 100 ml

    0.00292 g EDTA in 100 ml Polyvinylpolypyrrolidone (PVPP)

  • 2 mM Ascorbate, 100 ml

    0.03522 g ASC in 100 ml suspension solution

Dissolve PVP, EDTA and ascorbate in 80 ml of Na-P buffer (pH 7.0) and complete the volume to 100 ml with Na-P buffer. Assay Solutions

  • 50 mM K-PO4buffer (pH: 7.0), 100 ml
  • 5 mM Ascorbate, 30 ml

    Weigh 0.0264 g ascorbate, dissolve in 30 ml 50 mM K-PO4buffer.

  • 1 mM EDTA, 100 ml

    0.0292 g EDTA in 100 ml 50 mM K-PO4buffer (pH 7.0)

  • 1 mM H2O2, 100 ml

    Stock: 30% (w/v); MW=34.01 g/mol; d=1.11 kg/L

    Take 8.6 μl from stock and complete to 100 ml with 50 mM K-PO4buffer (pH 7.0).

b) Procedure

Extraction:

  • Weigh 0.5 g tissue, grind in a cold mortar and pestle with liquid nitrogen and suspend in 1.5 ml homogenization buffer.
  • Centrifuge the suspension at 14000 rpm for 30 min at 4 ∘C
  • Take the supernatant for the enzyme assay.

Assay Medium

  • 600 l 50 mM K-PO4buffer
  • 100 l mM EDTA
  • 100 l 5 mM ascorbate
  • 100 l sample
  • 100 l H2O2(0.1 mM) starts the reaction
Blank Sample
50 mM Na-P (pH 7) 700 l 600 l
1 mM EDTA 100 l 100 l
5mM Ascorbate 100 l 100 l
H2O2 100 l 100 l
Sample – 100 l

Record the reduction in ascorbate concentration by reading the absorbance at 290 nm continuously for 180 seconds. (Extinction Coefficient of Ascorbate (E) = 2.8 mM-1cm-1)

Literature references

Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) 867-880.

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