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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
superoxide, O2 - free radicals
hydroxyl radical, OH hydrogen peroxide,
H2O2 (Fentons
peroxyl, ROO
reaction)
alkoxyl, RO
hypochlorous acid,
hydroperoxyl, HO2 HClO
ozone, O3
singlet oxygen, 1O2
RNS (reactive nitrogen
species)
Free radicals Particals, which are not
nitrogen(II) oxide, NO . free radicals
nitrogen(IV) oxide, NO2 .
nitrosyl, NO +

nitrous acid, HONO


nitogen(III) oxide, N2O3
peroxynitrite, ONOO -
alkylperoxinitrite, ROONO
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
collagen synthesis E
dopamine to epinephrine localise in membranes
conversion produces
reduction agent hydroperoxides, which
Fe absorption are changes by GSHPx
antioxidant = reduction
O2 - OH , ROO, HO2
tocopheryl radical
regeneration
prooxidant
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
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 changes in fluidity


loss and permeability of
arising of reactive membranes
metabolites membranes integral
(aldehydes) enzymes are
influenced
Oxidative damage to
proteins
Damage Sequel

agregation, changes in: enzymes


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

saccharide ring mutation


cleveage translation mistakes
bases modification protoesynthesis
chain breakeage inhibition
Literature

tpek Stanislav a kol.: Antioxidanty a voln


radikly ve zdrav 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