Download oxidation, reduction, redox potential, citric acid cycle, respiratory

Biological oxidation
Key words: oxidation, reduction, redox potential, citric acid cycle, respiratory chain,
succinate dehydrogenase (EC 1.3.99.1), isocitrate dehydrogenace (EC 1.1.1.41),
malate dehydrogenace (EC 1.1.1.37), katalase (EC 1.11.1.6), peroxidase (POD - EC
1.11.1.7), peroxisomes, superoxide radical, superoxide dismutase (SOD - EC
1.15.1.1)
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. extract from potatoes
7. o-Tolidine (3,3´-dimethylbenzidine) 0.01 mol/L in glacial acetic acid !POISON!
8. Hydrogen peroxide 1 mol/L
9. Blood diluted with water in a ratio of 1:300
10. Paraffin oil
11. Potassium cyanide solution 0.02 mol/L !POISON!
Redox reactions are associated with the transfer of electrons and may be the source
of free energy for organisms. Oxidoreductases are enzymes with different
mechanisms of action, e.g oxidases (electron transfer), hydrogenases and
dehydrogenases (transfer of hydrogen atoms), oxygenases (transfer of oxygen
atoms) and hydroperoxidases (degradation of hydrogen peroxide).
Biological oxidation I – Citric acid cycle, respiratory chain and
oxidative phosphorylation
Citric acid cycle is metabolic connection of catabolic degradation of
saccharides, lipids and amino acids and its main aim is to produce reduced
coenzymes for energy production. Citric acid cycle is localized in matrix and inner
membrane of mitochondria and in one turn of cycle (processing 1 molecule of acetyl
coenzyme A) are produced 3 NADH, 1 FADH2, 1 GTP (immediately converted to
ATP) a 2 molecules of CO2.
Defects or abnormalities in citric acid cycle enzymes are probably incompatible
with living of organisms.
Oxidative phosphorylation (located on inner mitochondrial membrane) is a
process of ATP synthesis powered by proton gradient which is generated during
electron transfer from NADH and FADH2 to O2 in respiratory chain. The respiratory
chain is located on the inner mitochondrial membrane and it consists of a complex
system of membrane oxidoreductases (I. NADH – ubiquinone oxidoreductases, II.
succinate-ubichinonreduktasa, III. ubiquinone-cytochrome c-oxidoreductase and IV.
cytochrome c-oxidase) and mobile electron carriers (ubiquinone, Fe-S proteins and
cytochromes). Respiratory chain transfer protons and electrons. Proton gradient is
responsible for release of synthesized ATP from ATP synthases (oxidative
phosphorylation) and electrons reduce oxygen atoms to O2-. Oxygen anoints react
with protons in the mitochondrial matrix and form H2O (metabolic water).
Oxidative phosphorylation rate depends on need of ATP and sufficiency of
reduced coenzyme, O2, ADP and Pi are essential for this reaction.
The respiratory inhibitors include e.g. barbiturates and pesticide rotenone
(blocking of electron transfer between complex I and ubiquinone), antibiotic
Antimycine A (forming blockade between cytochrome c1 and complex III), carbon
monoxide,sulphates, cyanides and azides (blocking of electron transfer to oxygen in
cytochromeoxidase). Uncouplers of respiratory chain and oxidative phosphorylation
(e.g. 2,4-dinitrophenol, increased doses of the hormone thyroxine) cause protons
across inner mitochondrial membrane using other way than through ATP synthase.
ATP synthesis is blocked and chemiosmotic gradient forms heat. Organisms lacking
ATP increase degradation of nutrients. In case of hyperthyreoidism, associated with
overproduction of thyroxine, are observed weight lose and increase of body
temperature. Similar symptoms are typical for intoxication with 2,4-dinitrophenol.
Oxidative phosphorylation and respiratory chain inborn errors are mainly
manifested as disorders of organs with high energy turnover such are muscles
(myopathy), brain (encephalopathy) and optic nerve (blindness) - Leber's hereditary
optic neuropathy. Lactoacidosis is frequently manifested due to shift to anaerobic
metabolisms.
1. Evidence of the citric acid cycle dehydrogenases
Pripciple:
Oxidation (dehydrogenation) reactions of citric acid cycle are catalyzed i.e. by
isocitrate dehydrogenase, succinate dehydrogenase or malate dehydrogenase. They
are a source 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:
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 methyleneblue / leucoform (+ 0,01 V). Actual redox potential of system
depends according to Nernst’s –Peter’s 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 redoxpotential 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 2 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 room temperature (see table 1.).
c. The test-tube 4 insert in boiling water bath, boil 2 min, cool. Pour its contents into a
plastic test tube.
d. Follow the Table 1 (individual reagents add to all tubes in the sequence
determining by the rows in the table).
e. Shake all test tubes after addition every reagent.
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 37°C 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.
Notes:
Biological oxidation II – Reactive oxygen species
Reactive oxygen species (ROS) are high reactive atoms or molecules
containing free unpaired electrons. ROS include reactive molecules such are oxygen
radicals, hydrogen peroxide (H2O2), hydroxyl radical OH. and superoxide O2-.
Reactive nitrogen species (RNS) have similar characteristic and function as ROS.
The main source of ROS under physiological conditions is respiratory chain.
ROS are products of imperfect oxygen reduction. Fenton reaction is other possible
mechanism of ROS production. Metal ions (iron, copper) react with hydrogen
peroxide to form free radicals.
Fe2+ + H2O2 → Fe3+ + OH· + OHROS are highly reactive compounds damaging biological macromolecules.
They can cause breaks and mutations of DNA, decreased enzyme activity and
damaging of ion channels in cell membranes. ROS are responsible for membrane
lipids peroxidation contributing to changed permeability and damage of cell
membranes.
ROS are associated with pathogenesis of several diseases and pathological
conditions e.g. tissue damage after reperfusion of ischemic tissues, artherosclerosis,
neurodegenerative processes (Alzheimer disease).
RNS and ROS have important roles in many physiological mechanisms
especially in cellular immunity and intercellular signalization.
Excess of ROS is removed from the body by several enzymes, including
superoxide dismutase, catalase and peroxidase.
Superoxide dismutase catalyzes the conversion of free radical superoxide to
hydrogen peroxide, which is further degraded by catalase and peroxidase.
-
2O2 + H2 → H2O2 + O2
Catalase degradates hydrogen peroxide into oxygen and water
H2O2 → 2 H2O + O2
Peroxidase needs electron donor for its action. Typical example of compounds,
supplying electrons for the reduction of H2O2 by peroxidase, is gluthathione which is
oxidized by the enzyme gluthathione peroxidase.
2. Detection of peroxidase activity
Peroxidase (POD) catalyzes the reduction of hydrogen peroxide to water in
the present oxidation of an appropriate reactant, in this case, o-tolidine. The oxidation
product is colored in cyan:
POD
H2O2 + AH2 ―—―→ 2 H2O + A
Similar reactions are used for spectrophotometric determination of the concentration
of various metabolites (eg, glucose, uric acid, cholesterol) in serum or urine, where
hydrogen peroxide is produced as a by-product of the appropriate enzyme reaction.
The quantity proportional to the concentration of the analyte is determined in the
subsequent peroxidase reaction. Added chromogen (AH2) is oxidized during the
course of the colored compound suitable for photometric determination.
Procedure:
a. Within four calibrated tubes, prepare the incubation mixture, follow the order of the
rows in Table 3
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°
Isolated extract
from potatoes (ml)
Boiling in hot
water (2 min)
o-Tolidine
Hydrogen peroxide
(ml)
Distilled water (ml)
1
2
3
4
1
1
1
-
4 drops
yes
4 drops
4 drops
4 drops
0.2
-
0.2
-
0.5
0.2
1
Colour
d. Explain the course of individual reactions. Write down the results in the table.
Notes:
3. Detection of catalase activity and pseudoperoxidase reaction
Catalase is a heme enzyme present in most aerobic cells. In the human
organism is its high activity in hepatocytes and erythrocytes. At the subcellular level
is localized predominantly in the peroxisomes (contain enzymes that produce
hydrogen peroxide).
Removal of hydrogen peroxide by peroxidase and catalase is defined e.g. in
the prevention of the Fenton reaction.
This task is demonstrated catalase activity of the blood that affects the development
of oxygen. Catalase is inhibited by cyanide, like cytochromeoxidase.
In blood, in addition to the catalase and peroxidase activity, we can also
demonstrate pseudoperoxidase activity . It is linked to the presence of ferrous ions in
the blood hemoglobin. Fe2+ catalyzes the decomposition of hydrogen peroxide. The
result is the reduction of peroxide to water and the oxidation of o-tolidine to bluegreen oxidation product. This reaction is not catalyzed by the enzyme and couldn’t be
inactivated by boiling. Pseudoperoxidase reaction is used for the detection of blood in
urine
Procedure:
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°
Blood diluted with water
Boiling in hot water (2 min)
Potassium cyanide
Hydrogen peroxide
Generation of oxygen
o-Tolidine
Colour
1
2
3
1 ml
1 ml
1 ml
-
yes
0.2 ml
0.2 ml
0.2 ml
0.2 ml
2 drops
2 drops
2 drops
e. Explain the course of individual reactions. Write down the results in the table.
Notes: