OXIDOREDUCTASES

Key words: oxidation, reduction, redox potential, citric acid cycle, respiratory chain,
succinate dehydrogenase (EC 1.3.99.1), cytochromoxidase (EC 1.9.3.1), cytochromes,
inhibition of enzymes, competitive inhibition, enzymes, catalase, peroxidase, peroxisomes,
superoxide radical, superoxide dismutase, glutathione peroxidase, hypoxanthine, xanthine,
uric acid, xanthine oxidase

Reagents:
1. Bovine heart
2. Phosphate buffer 0.1 mol/l, pH 7,4
3. Methylene blue solution 0.1g/l
4. Sodium succinate solution 0.1 mol/l in the phosphate buffer
5. Sodium malonate solution 1 mol/l in the phosphate buffer
6. Potassium cyanide solution 0.02 mol/l !POISON!
7. 1,4-phenylenediamine solution 0.01 mol/l
8. Paraffin oil
9. o-Tolidine (3,3´-dimethylbenzidine) 0.01 mol/l in glacial acetic acid
10. Hydrogen peroxide 1 mol/l
11. Blood diluted with water in a ratio of 1 : 300
12.Potassium cyanide !POISON! 0.02 mol/l
13. Fresh milk
14. Boiled milk
15.Formaldehyde 0,4%
16. enzyme extract
17. isolated extract

1.Succinate dehydrogenase

Pripciple:
Oxidation (dehydrogenation) reactions of citric acid cycle are catalyzed i.e. by
isocitrate dehydrogenase, succinate dehydrogenase or malate dehydrogenase. They are
funds of reduced coenzyme NADH and FADH2, which transport hydrogen atoms into
respiratory chain. They are further transport like protons and electrons through single
components of respiratory chain to a final acceptor - molecular oxygen (O2).
In this model attempt succinate dehydrogenase is tested. Sodium succinate is used like a
substrate, methylene blue serves like the acceptor of hydrogen atoms again (see exercise 2.):

Ox / Red Eo´ (V)

fumarate / succinate + 0.03

methylene blue / leucoform + 0.01

O2 / H2O2 + 0.68

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 In this model, dehydrogenation proceeds spontaneously in spite of standard redox potential
of system fumarate / succinate is higher (+ 0,03 V) than redox potential of system methylene
blue / leucoform (+ 0,01 V). Actual redox potential of system depends according to Nernstś –
Peterś equation not only on Eo but also on the concentrations of reduced and oxidized
components of both systems as well. In this case, the concentration of the reduced form
(succinate) of one’s redox system is higher, and in the second system, the concentration of
oxidized form (methylene blue) is higher at the same time. This fact influences actual redox
potential values so, that the oxidation of succinate by means of methylene blue proceeds
spontaneously.
This model experiment demonstrates also the competitive inhibition of succinate
dehydrogenase by malonate. It serves as competitive inhibitor of this enzyme thanks its
structural similarity with succinate.

Procedure:
a. Pipette 1 ml of enzyme extract into four test tubes 1-4.
b. Pipette 0.5 ml of sodium malonate solution into test tubes 3 and let incubate 10 min at
laboratory temperature (see table 1.).
c. The test-tube 4 insert in a warm water bath, boil 2 min, cool.
d. From the 6th line in the table 1. (buffer), there is necessary to unconditionally follow the
single steps of procedure according to lines for all test-tubes all at once.
e. Shake all test tubes after addition every reagent.

Table 1.
Tube No 1 2 3 4
Enzyme extract (ml) 1 1 1 1
Sodium malonate (ml) - - 0.5 -
Incubation 10 min - - yes -
Boiling 2 min - - - yes
Buffer (ml) 1.0 0.5 - 0.5
Sodium succinate (ml) - 0.5 0.5 0.5
Methylene blue (ml) 0.5 0.5 0.5 0.5
Colouring

f. Add slowly into every test-tubes up its side paraffin oil so, to form an approximately
5 mm high layer for to be preserving anaerobic conditions.
g. Incubate 30 min at 37oC and watch gradual discolouration of methylene blue.
h. After discoloration pertinent test-tubes shake and let react again.
i. Resulting colouring inscribe in table 1.

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2. Cytochrome oxidase

Principle:
Cytochrome oxidase oxidises reduced cytochrome c. It is the terminal part of the
respiratory chain in mitochondria: four electrons are transferred from cytochrome c to
molecular oxygen; the oxygen anions then combine with protons to form water. Cytochrome
oxidase is an enzyme complex bound to the inner mitochondrial membrane. The cytochrome
oxidase complex contains haeme iron as prosthetic groups, oxidation number of iron changes
during redox reaction from 3+ to 2+and vice versa.
Cyanide, nitrogen (II) oxide and carbon monoxide are inhibitors of the oxidised or reduced
form of the enzyme, respectively. Ions Fe3+, possessing higher affinity to CN- than Fe2+, form
stabile complex with cyanide anions.
In this experiment, 1,4-phenylenediamine serves as an electron donor for the reduction of
cytochrome c. The cytochrome c is present in bovine heart extract together with the
cytochrome oxidase, which is very specific and can accept electrons only from reduced
cytochrome c. The oxidation product of 1,4-phenylenediamine yields with another molecule
of this compound a red-brown dye. Since the oxidation of 1,4-phenylenediamine is coupled
with the action of cytochrome oxidase in this experimental arrangement, it allows a
qualitative estimation of this enzymatic activity. The inhibition of cytochrome c oxidase with
cyanide will be also demonstrated.

Ox / Red Eo´ (V)

HN=C6H4=NH / H2N-C6H4-NH2

cytochrom c
Fe3+/ Fe2+ +0.26

cytochromoxidasa
Fe3+/ Fe2+ +0.29

1/2 O2 / O2- +0.82

Procedure:
a. Into four graduated tubes prepare reaction mixture following table 2.
b. Boil the tube 4 in a warm water bath for 2 min, cool.
c. Follow the single steps of procedure according to lines for all test tubes all at once.
d. Use doser to add sodium cyanide and 1,4-phenylenediamine.
e. Shake all test tubes after addition every reagent.

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Table 2.
Tube No 1 2 3 4
Enzyme extract (ml) 0.5 0.5 0.5 0.5
Distilled water (ml) 2.5 2.5 2.5 2.5
Boiling 2 min - - - yes
Potassium cyanide (ml) - - 0.2 -
1,4-phenylenediamine (ml) - 0.2 0.2 0.2
Colouring

f. Incubate all test tubes 15 min at 37oC and watch the change in colouring of reaction
mixture.
g. Resulting colouring inscribe in table 2.

2.Isolation of peroxidase

Principle:
Peroxidases belong to heme or non heme enzymes occurring in many plant and animal
tissues. Peroxidases catalyze the decomposition of hydrogen peroxide. In human,
glutathione peroxidase protects against damaging effects of peroxide, which is produced
when fats are oxidized. Selenium is the trace element essential for the activity of this
enzyme. Peroxidase used in our experiments will be isolated from potatoes.

Procedure:
a. Place 20 g of potatoes in 50 ml of distilled water.
b. Grind with tissue homogenizer until no pieces are visible.
c. Filtrate the homogenate through the three times folded gauze.
d. 5 ml of filtrate (the volume is measured in graduated cylinder) will be used in next
exercise.

A.Detection of peroxidase activity

Principle:
The enzyme peroxidase catalyzes the oxidation of many substrates by hydrogen
peroxide.

POD
H2O2 + AH2 ―—―→ 2 H 2O + A

A chromogenic compound, such as o-tolidine used in this exercise, can be oxidised to

form a coloured product.

Procedure:

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a. Prepare incubation mixtures as described in the table.
b. The test tube N° 2 is incubated for 2 min in boiling water bath.
c. The formation of coloured product indicates the presence of peroxidase activity.

Test tube N° 1 2 3 4
Isolated -
extract 1 ml 1 ml 1 ml
o-Tolidine 4 drops 4 drops 4 drops 4 drops
Hydrogen -
peroxide 0.5 ml 0.5 ml 0.5 ml
Distilled - -
water 0.5 ml 1 ml
Colour
d. Explain the course of individual reactions. Write down the results in the table.

B.Detection of catalase activity and pseudoperoxidase reaction

Principle:
Catalase is a heme enzyme, which may constitute as much as 40% of the total protein
contained in the organelles known as peroxisomes. Peroxisomes are abundant in liver
and many other tissues. They contain D-amino acid oxidase, which produces hydrogen
peroxide, however, catalase, in the same organelle, promotes the efficient destruction of
hydrogen peroxide. The enzyme decomposes H2O2 to form water and free molecular
oxygen. The reaction is shown by the following equation.

Catalase
2H2O2 ―――→ 2H2O + O2

Catalase also decomposes H2O2 formed from superoxide radical by the action of
superoxide dismutase. Toxic superoxide radical is produced as a by-product of many
oxidative reactions. All aerobic cells contain enzyme, superoxide dismutase, that
detoxify O2‾ by catalysing the dismutation reaction:

2O2‾ + 2H+ → H2O2 + O2

In this exercise the catalase activity will be demonstrated in blood. The generation of
oxygen indicates the activity of catalase. Catalytic activity of catalase is inhibited by
cyanide ions.
Peroxidase presents in blood catalyses the oxidation of o-tolidine to form a coloured
product. Heating inactivates the enzyme. However, haemoglobin and its derivatives also
catalyse the reaction between hydrogen peroxide and a chromogenic compound. The
peroxidase activity in haemoglobin (this activity is also called pseudoperoxidase
activity) is not affected by heating. The peroxidase test is a common screening test for
blood in urine and faeces.

Procedure:

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a. Prepare incubation mixtures as described in the table.
b. The test tube number 2 is incubated for 2 min in boiling water bath.
c. The generation of oxygen proves the activity of catalase.
d. The formation of coloured product indicates the presence of peroxidase activity.

Test tube N° 1 2 3
Blood diluted with 1 1 1
water (ml)
Potassium cyanide (ml) - - 0.2
Hydrogen peroxide (ml) 0,5 0,5 0,5
o-Tolidine 4 drops 4 drops 4 drops
Generation of oxygen
Colour
e. Explain the course of individual reactions. Write down the results in the table.

C.Xanthine oxidase

Principle:
Xanthine oxidase (EC 1.2.3.2), a molybdenum and iron-containing flavoprotein,
oxidises hypoxanthine to xanthine and then to urate. In humans, urate is the final
product of purine degradation and is excreted in the urine. Molecular oxygen, the
oxidant in both reactions, is reduced to H2O2, which is decomposed to H2O and O2 by the
action of catalase. Xanthine oxidase is relatively unspecific. Oxidation of aliphatic
aldehydes can be also demonstrated. Xanthine oxidase is not inhibited by cyanide ions
but is destroyed by boiling.
We will demonstrate the activity of xanthine oxidase in milk. Autooxidable day,
methylene blue, will serve as the acceptor of hydrogen atoms. The oxidised methylene
blue has a blue colour, reduction changes it to the colourless leucoform. Methylene blue
turns blue again when the reaction mixture is shaken.

Procedure:
a. Prepare reaction mixture as follows:
b. Immediately after mixing, overlay reaction mixtures with paraffin oil. The oil
prevents access to air oxygen and autooxidation.

Tube No 1 2
Fresh milk (ml) 2.5 -
Boiled milk (ml) - 2.5
Methylene blue (ml) 0.5 0.5
Formaldehyde 2 drops 2 drops
Paraffin oil (ml) 1.0 1.0
Results:
c. Incubate the test tubes in a water bath at 37˚ C. Observe the reaction every two
minutes, until the contents of tube No 1 is colourless.
d. Shake the tube No 1: the contents turn blue again.
e. After further incubation in water bath the methylene blue looses its colour again.
f. Explain the differences in the results obtained. Write down the results in the table.

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