OXIDATION BIOLOGY

(OXIDATIVE STRESS)
 Free radical

 particles with an unpaired electron spinning around

the nucleus. (can be atom, ions, molecule).
 tend to reach equilibrium, plucks an electron from the
nearest intact molecule.
ROS (reactive oxygen species)
Free radicals Particals, which are not free
superoxide, O2 · - radicals
hydroxyl radical, OH · hydrogen peroxide, H2O2
peroxyl, ROO · (Fenton´s reaction)
alkoxyl, RO · hypochlorous acid, HClO
hydroperoxyl, HO2 · ozone, O3
singlet oxygen, 1O2
RNS (reactive nitrogen species)
Free radicals Particals, which are not free
nitrogen(II) oxide, NO . radicals
nitrosyl, NO +
nitrogen(IV) oxide, NO2 .
nitrous acid, HONO
nitogen(III) oxide, N2O3
peroxynitrite, ONOO -
alkylperoxinitrite, ROONO
OXIDATIVE STRESS
OXIDATIVE STRESS
Mechanism of radical reactions
Radicals are highly reactive species

Three distinc steps

 initiation (homolytic covalent bonds cleavage)

 propagation (chain propagation)
 termination
The main sources of free radicals
membranes enzymes and/or coenzymes with flavine
structures, hem coenzymes, enzymes containing Cu
atom in an active site
1. respiratory chain mitochondria : mainly superoxide
and then H2O2
 approx 1- 4% O2 entres into resp. chain (mainly
complexes I a III)
The main sources of free radicals II
2. Endoplasmic reticulum
superoxide creation (by cytochrome P- 450)
3. special cells (leukocytes)
superoxide creation by NADP-oxidas
4. hemoglobin to methemoglobin oxidation
(erytrocyte is „full“ of antioxidants)
Free radicals physiological function
Used by oxides a oxygenes
 cytochromoxidase (toxic intermediates, H2O2 and
superoxide, are bound to an enzymu)

 monoxygenases - activate O2 in liver ER or in adrenal

gland mitochondria ; hydroxylation
Free radicals physiological function II
ROS a RNS against bacteria
 enzyme complex NADPH-oxidase of leukocytes

 myeloperoxidase - catalysis of the following reaction

H2O2 + Cl- + H+ = HClO + H2O
Free radicals physiological function III
 Signal molecules
first messenger  second messenger  information net
This info net function is affected by the redox state of cells
 redox state : antioxidant capacity, reduction equivalent
availlability, RONS rate
 ROS: second messenger
 Antioxidant defence system

3 levels

inhibition of production the abundance of RONS

capture of radicals (scavengers, trappers, quenchers)

correction mechanism of destroyed biomoleculs

Antioxidants and scavengers review
1. Endogennous antioxidants

 enzymes (cytochrome c,SOD, GSHPx, catalase)

 nonenzymatic
- fixed in membranes ( -tocopherol, -caroten,
coenzym Q 10)
- out of membranes (ascorbate, transferrin,
bilirubin)
Antioxidants and scavengers review II
2. Exogennous antioxidants

 FR scavengers
 trace elements
 drugs and compounds influence to FR metabolism
 Superoxid dismutase

2O2. - + 2H+  H2O2 + O2

SOD - is present in all oxygen-metabolizing cells,

different cofactors (metals)
an inducible in case of superoxide overproduction
 Superoxid dismutase
Mn 2+ SOD (SOD1)
tetramer
matrix mitochondria
lower stability then Cu, Zn - SOD
 Superoxid dismutase
Cu 2+/Zn 2+ SOD (SOD 2)

dimer, Cu = redox centr

cytosol, intermitochondrial space
hepatocyt, brain, erytrocyte
high stability, catalysation at pH 4,5-9,5
 Glutathion peroxidases

elimination of intracellular hydroperoxides and H2O2

2 GSH + ROOH  GSSH + H2O + ROH
 cytosolic GSH - glutathionperoxidasa (EC 1.11.1.9,
cGPx)
 extracelullar GSH - glutathionperoxidasa (eGSHPx)
 phospholipidhydroperoxide GSH - peroxidase (EC
1.11.1.12, PHGPx)
• transferrin
• ferritin

• haptoglobin
• hemopexin

• albumin
 Low-molecule endogennous antioxidats I
Ascorbate (vitamin C) Alfa-tocopherol a vitamin E
collagen synthesis localise in membranes
dopamine to epinephrine produces hydroperoxides,
conversion which are changes by
reduction agent GSHPx
Fe absorption
antioxidant = reduction O2 · -
OH ·, ROO·, HO2 ·
tocopheryl radical
regeneration
prooxidant
Ascorbic acid and its metabolites
 Low-molecule endogennous antioxidats II

 ubiquinone (coenzyme Q)
electron carrier in respisratory chain
co-operates with tocopheryl

 carotenoides, -caroten, vitamin A

removing the radicals from lipids
 Low-molecule endogennous antioxidats III

 glutathione (GSH, GSSG)

in all mammalian cells (1-10 mmol/l)
important redox buffer
2 GSH  GSSG + 2e- + 2H+
ROS elimination, stabilisation in reduction form ( SH-
groups, tocopheryl and ascorbate regeneration)
substrate of glutathione peroxidases
 Low-molecule endogennous antioxidats IV

 Lipoic acid (lipoate)

PDH cofactor
tocopheryl and ascorbate regeneration
 melatonin
lipophilic ; hydroxyl radicals scavenger
Low-molecule endogennous antioxidats V

 uric acid (urates)

 bilirubin

 flavonoids
Trace elements influence to FR
metabolism
Selenium
influence to vitamin E resorption, part of
selenoproteins
 of Se = insufficient immun. respons,
erytrocytes hemolysis, methemoglobin synthesis

Zinc
cell membrane stabilisation
Fe antagonist
 Oxidative damage to lipid
Damage Sequel

 unsaturated bonds loss  changes in fluidity and

 arising of reactive permeability of
metabolites (aldehydes) membranes
 membranes integral
enzymes are influenced
The peroxidation of linoleic acid
Oxidative damage to proteins
Damage Sequel

 agregation, fragmentation  changes in: enzymes

and cleveage activity, ions transport
 reaction with hem iron ion  proteolysis
 functional group
modification
 Oxidative damage to DNA
Damage Sequal

 saccharide ring cleveage  mutation

 bases modification  translation mistakes
 chain breakeage  protoesynthesis
inhibition
 Literature

Štípek Stanislav a kol.: Antioxidanty a volné

radikály ve zdraví a nemoci, Grada, 2000

Free radicals and antioxidant protocols

edited by Armstrong D., Methods in
Molecular biology, volume 108, HUMANA
PRESS, Toronto, New Yersey, 1998