Antioxidant enzymes

Catalase is a ubiquitous enzyme found in all known organisms. In eukaryotic cells, it is present in peroxisomes.

This enzyme contains a heme in its active site responsible for its catalytic activity:

two mole of H2O2 , are transformed into two moles of H2O and one mole of O2.

As for superoxide dismutase, this enzyme can be induced in some conditions by exposure of cells or organisms to oxidative stresses .

Catalase

The activity of catalase (EC 1.11.1.6) was determined according to Aebi (1974).

Enzyme extract (100 µl) was added to 2.9 ml of a reaction mixture containing 20 mM H2O2 and 50 mM sodium phosphate buffer (pH 7.0).

The activity of CAT was measured by monitoring the reduction in the absorbance at 240 nm as a result of H2O2 consumption.

The amount of consumed H2O2 was calculated by using a molar extinction coefficient of 0.04 cm2 µmol-

1. CAT activity was expressed as units min-1 mg-1 protein. One unit of enzyme activity was defined as the decomposition of 1 µmol of H2O2 / min.

Assay catalase activity

peroxidase

Peroxidases (EC number 1.11.1.x) are a large family of enzymes that typically catalyze a reaction of the form:

ROOR' + electron donor (2 e-) + 2H+ → ROH + R'OH

For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or redox-active cysteine or selenocysteine residues.

The nature of the electron donor is very dependent on the structure of the enzyme.

For example, horseradish peroxidase can use a variety of organic compounds as electron donors and acceptors. Horseradish peroxidase has an accessible active site, and many compounds can reach the site of the reaction.

For an enzyme such as cytochrome c peroxidase, the compounds that donate electrons are very specific, because there is a very closed active site.

Glutathione peroxidases use glutathione as an electron donor and are active with both hydrogen peroxide and organic hydroperoxide substrates

The activity of POX (EC1.11.1.7) was assayed by the method of Hammerschmidt et al. (1982).

The reaction mixture (3 ml) consisted of 0.25 % (v/v) guaiacol in 10 mM sodium phosphate buffer (pH= 6 containing 10 mM hydrogen peroxide H2O2).

Volume of 100 µl of the crude enzyme extract was added to initiate the reaction which was measured at 470 nm per min.

One international (IU) of enzyme activity was expressed as Δ OD= 0.01. POX activity expressed as units .min-1.mg-1 protein

Assay of peroxidase activity

H2O2 +

Superoxide dismutases

Superoxide dismutases are metalloenzymes that catalyze the reduction of superoxide anion (O2

-•) to the less reactive H2O2.

Three distinct enzymes have been described with the same kinetic properties:

one containing iron in its active site found in prokaryotes,

one with manganese (MnSOD) in prokaryotes and eukaryotic mitochondria, and one with copper and zinc (Cu/ Zn-SOD) in the cytoplasm of eukaryotic cells.

The activity of SOD (EC 1.15.1.1) was assayed by the method of Beauchamp and Fridovich (1971) by measuring its ability of enzyme to inhibit the photochemical reduction of nitrbluetetrazolium (NBT).

A reaction mixture (3 ml) containing 40 mM phosphate buffer (pH=7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA and 100 µl of the crude enzyme extract was shaken and placed 30 cm below light source consisting of 15 W fluorescent lamp.

The absorbance was recorded at 560 nm. The activity of SOD was expressed at unit mg-1 protein. One unit of SOD activity is the amount of protein required to inhibit 50% initial reduction of NBT under light. Enzyme activity was expressed as (IU. mg-1Protein).

Assay of superoxide dismutase.

One unit of enzyme activity was determined as the amount of the enzyme to reach an inhibition of 50% NBT reduction rate by monitoring the absorbance at 560 nm with a spectrophotometer .

This indirect assay is comprised of several reactions:

the photochemically excited riboflavin was first reduced by methionine into a semiquinone, which donated an electron to oxygen to form the superoxide source.

The superoxide readily converted NBT into a purple formazan product.

Nitro Blue Tetrazolium

coloured formazan

Superoxide dismutase (SOD) are metalloenzymes that catalyze the dismutation of superoxide radical into hydrogen peroxide (H2O2) + molecular oxygen (O2) and consequently provide an important defense mechanism against superoxide radical toxicity (1).

To determine SOD activity, several direct and indirect methods have been developed. A common and convenient indirect method utilizes nitroblue tetrazolium (NBT) conversion to NBT-diformazan (formazan dye) via superoxide radical. However, there are several disadvantages to the NBT method, such as poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase. Though cytochrome C is also commonly used for SOD activity detection, its reactivity with superoxide is too high to determine low levels of SOD activity.

Assay Principle:

To determine SOD activity, several direct and indirect methods have been developed. A common and convenient indirect method utilizes nitroblue tetrazolium (NBT) conversion to NBT-diformazan (formazan dye) via superoxide radical. However, there are several disadvantages to the NBT method, such as poor water solubility of the formazan dye and the interaction with the reduced form of xanthine oxidase. Though cytochrome C is also commonly used for SOD activity detection, its reactivity with superoxide is too high to determine low levels of SOD activity.