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2.  Definition:
Free radical is a molecule or molecular
fragment that contains one or more
unpaired electrons in its outer orbits.

4.  Free radicals conventionally represented by super
script dot R•
 Characteristics of free radicals :
1 )Tendency of free radicals to acquire electrons
from other substance makes it more reactive.
2 )Short life span
3 )Generation of new free radicals by chain reaction.
4 )Damage to various tissues.

5.  Partial reduction of oxygen leads to
formation of free radicals called as reactive
oxygen species .The following are members
of this group.
Super oxide anion radical (O2 · )
Hydroperoxy radicals ( HOO· )
Hydroxyl radical ( OH· )
Lipid peroxyl radical ( ROO· )
Nitric oxide ( NO· ) , Peroxy nitrite (ONOO · )
H2O2 , singlet oxygen (are not free radicals)

6.  Free radicles are generated in oxidative
metabolism due to leak of electrons .
 Flavoprotein linked oxidases like xanthine
oxidase , L α amino acid oxidase .
 Super oxide is formed by autooxidation of
hemoglobin to methemoglobin
(approximately 3 % of the Hb has been
calculated to autooxidise per day )

7.  Cyclooxygenase & lipoxygenase reactions in
metabolism of eicosanoids.
 NADPH oxidase system of inflammatory cells
by process of respiratory burst during phase
of phagocytosis.
 Free iron causes increased production of free
radicals .

8.  Free radicals are formed cytochrome P450
reductase enzyme complex durinrg
metabolism of xenobiotics .
 β oxidation of very long chain fatty acids in
peroxisomes produces H2O2 .

9.  Transfer of 4 electrons from reduced
cytochrome C to molecular oxygen assisted
by cytochrome oxidase
 Transfer of 4 electrons lead to safe product
H2O .
 Site of electron escape appears to be
ubiquinone & cytochrome C .

10.  Cytochrome C oxidase does not release
partially reduced intermediates , this crucial
criterion meets by holding O2 tightly
between Fe & Cu atoms .

11.  Although Cyt C oxidase & other protiens that
reduce O2 are remarkably successful in not
releasing intermediates , small amounts of
super oxide & peroxyl radicals are
unavoidably formed.
 About 1-4 % of oxygen taken up in the body is
converted to free radical .

13.  Flavoprotien linked oxidases
1 ) Xanthine oxidase ,
2) L α amino acid oxidase ,
3 ) Aldehyde dehydrogenase .
 Reduction of isoalloxazine ring of flavin
nucleotides takes place in 2 steps via a
semiquinone ( free radical ) intermediate.

14. xanthine oxidase
Hypoxanthine xanthine
O2 O2 ·
acetaldehyde dehydrogenase
Acetaldehyde acetate
O2 O2 ·

16.  NADPH oxidase inflammatory cell produce
supere oxide anion by a process of respiratory
burst during phagocytosis.
 This is the deliberate production of free
radicals by the body .

17. activation of inflammatory cell
drastic increase in consumption of oxygen
(respiratory burst )
10% of oxygen uptake by macrophage is used
for free radical generation .

19.  In chronic granulomatous disease the NADPH
oxidase is absent in macrophages &
neutrophils .
 Streptococci & pneumococci themselves
produce H2O2 therefore they are destroyed
by myeloperoxidase system .

20.  Staphylococci being catalase + ve can
detoxify H2O2 in the macrophages & they are
not destroyed .
 Hence recurrent pyogenic infections by
staphylococci are common in CGD .

21.  Prodstaglandin H synthase & lipooxygenase
enzyme catalysed reactions produce free
radicals , by producing peroxide .
 Macrophages produce NO from arginine by
enzyme nitric oxide synthase , this is also an
important anti bacterial mechanism .

22.  Super oxide ion can release iron from ferritin .

23.  The capacity to produce tissue damage by
H2O2 is minimal because this is not a free
radical . But in the presence of free iron H2O2
can generate hydroxyl free radical (OH
·)which is highly reactive.

25.  Ionising radiation damages tissues by
producing hydroxylradical , H2O2 ,super
oxide anion .
 Light of appropriate wave length can cause
photolysis of oxygen to produce singlet
oxygen .
 Cigarette smoking contains high
concentrations of free radicals.
 Other toxic compounds CCl4 drugs &
inhalation of air pollutants will increase free
radical production .

26.  Polyunsaturated fatty acids present in cell
membranes are destroyed by peroxidation.
 This occurs by three phases.
1 )intiation phase
2 )prolangation phase
3 )termination phase

27.  Production of carbon centered free radical R·
( or ) ROO· (lipid peroxide radical )
1 )RH +OH· R· + H2O
metal ion
2 )ROOH ROO· + H+
R· , ROO· degraded to malon dialdehyde . It is
estimated as an indicator of fatty acid break
down by free radical .

28.  Carbon centered radical rapidly reacts with
molecular oxygen forms peroxyl radical
(ROO· ) which can attack another PUFA .
R· + O2 ROO·
ROO· + RH ROOH + R·
 One free radical generates another free
radical in the neighbouring molecule a chain
reaction (or) propagation is intiated .

29.  The above reactions would proceed
unchecked till a peroxyl radical reacts with
another peroxyl radical to form inactive
products .
ROO· + ROO· RO- -OR+O2
R· + R· R - - R
ROO· + R· RO- -OR

30.  Super oxide dismutase
 Catalase
 Glutathione peroxidase
 Cytochrome oxidase

31.  Chief amongst the enzymes that defense
against ROS is super oxide dismutase .
 Super oxide dismutase is present in all major
aerobic tissues .
 Eukaryote contains 2 forms of this enzyme,
1 ) Copper Zinc dependent cytosolic enzyme
2 ) manganese containing mitochondrial
enzyme .

34.  The active site of cytosolic enzyme in
eukaryotes contains a copper ion & Zinc ion
coordinated to the side chain of a histidine
residue .
 The negatively charged superoxide is guided
electrostatically to a very positively charged
catlytic site at the bottom of the channel .

36.  H2O2 formed by SOD & by other processes is
scavenged by catalase ( a ubiquitous heme
protein that catalyze the dismutation of
H2O2 into H2O & O2.)
 Catalase is found in blood bone marrow
mucous membranes , liver & kidney .
 SOD & catalase are remarkably efficient ,
performing their reactions at or near the
diffusion limited rate.

37.  The Kcat / Km ratio of enzyme super oxide
dismutase is 7x 10 9 enzymes that have high
K cat / K m ratio at the uper limits have
attained kinetic perfection.
 Their catalytic velocity restricted only by the
rate at which they encounter the substrate in
the solution .

38.  For catalytically perfect enzymes , every
encounter between enzyme & substrate is
productive .
 Any rate in catalytic rate can come only by
decreasing the diffusion .
 Circe effect : In this case the electrostatic
attractive forces on the enzyme entice the
substrate to the active site .

39.  Catalase decreases the free energy of
activation ∆G 1 of H2O2.
 In the absence of catalase ∆G1 free energy of
activation is 18Kcal / mol where as in the
presence of catalase 7 Kcal / mol .
 K cat / Km value of catalase is 4 X 107.

40.  Catalase is a heme protein containing 4 heme
groups .
 In addition to possessing peroxidase activity ,
it is able to use one molecule of H2O2 as a
substrate electron donor & another molecule
of H2O2 as oxidant or electron acceptor .
catalase
2H2O2 2H2o+O2

42.  This enzyme is remarkable in containing a
modified aminoacid selenocystein at its
active site in which selenium has replaced the
sulphur .
 The enzyme catalyzes the destruction of
H2O2 & lipid hydroperoxides by reduced
glutathione , protecting the membrane lipids
& hemoglobin against oxidation by peroxides
.

46.  Vitamin E : lipid soluble , chain breaking
antioxidant.
 βcarotene & its anologues (lycopene & retinyl
stearate ): lipid soluble radical scavenger & singlet
oxygen quencher .
 Coenzyme Q : may acts as antioxidant in addition to
its major role in energy metabolism .

47.  Transferrin : binds ferric ions ( 2 per mole of
protein )
 Lactoferrin : binds ferric ions at low pH ( 2 per
mole of protein )
 Haptoglobins : binds hemoglobin
 Albumin : binds copper , heme , scavenges
OH.
 Ceruloplasmin : ferrooxidase activity –
stoichiometricO2 scavenging ,binds copper
ions utilizes H2O2 for reoxidation of copper .

48.  Ascorbic acid OH radical scavenger
 Bilirubin : scavenges peroxyl radicals, open
chain tetra pyrroles are effective singlet
oxygen quenchers .
 Urate : radical scavenger & metal binder
 Mucus : scavenges OH radicals
 Glucose : OH radical scavenger .

49.  Water soluble : urate , ascorbates ,
thiols ,bilirubin,
flavanoids.
 Lipid soluble : tocopherol ,
ubiquinol 10 ,
β carotene .
 Urate & vitamin E acts in lipid phase to trap
ROO· radicals .

50.  Preventive antioxidants reduces the rate of
chain intiation .
 Preventive antioxidants include
Catalase , peroxidases ,
Ceruloplasmin , transferrin , albumin.
 EDTA , DTPA acts anti xidants by chelating
metal ions .

51.  Vitamin E is most important natyral
antioxidant .
 Vitamin E apear to be the 1st line defense
against peroxidation of PUFA contained in
cellular & subcellular membrane
phospholipids .
 The phospholipids of mitochondria,
endoplasmic reticulum & plasmamembranes
possess affinities for αtocoferol & vitamin
appears to concentrate at these sites.

52.  The tocopherools acts as antioxidants by
breaking free radical chain reactions as a
result of their ability to tranfer a phenolic
hydrogen to peroxyl free radical of a
peroxidized PUFA .
 The phenoxy free radical may react with
vitaminC to regenerate tocopherol or it react
with further peroxyl free radicl so that the
chromane ring & the side chain are oxidized
to the non free radical product.

53. ROO· +TocOH ROOH +TocO·
ROO· +TocO· ROOH + non free
radical product

54. ROO· +TocOH ROOH +TocO·
ROO· +TocO· ROOH + non free
radical product

56.  The antioxidant action of vitamin E is
effective at high oxygen concentrations , & it
is concentrated in lipid structures exposed to
highO2 partial pressures such as the
erythrocyte membrane , membranes of
respiratory tree & the retina.

57.  Glutathione peroxidase contains
Selenium, provides a second line of defense
against hydroperoxides.
 Tocopherol & selenium reinforce each other
in their action against lipid peroxides.
 Selenium is required for normal pancreatic
function thus promoting absorption of lipids
& vit E
 Vitamin E reduces selenium requirement by
preventing loss of selenium from the body or
maintaining it in an active form .

58.  Carotenoids are capble of quenching singlet
molecular oxygen .
 Carotenoids like lycopene ,β carotene , are
important biological molecules that can
inactivate electronically excited molecules by
process called quenching.
 βcarotene & related compounds can acts as
chain breaking antioxidant.
 Can acta as preventive antioxidant by
decreasing the formation of methyl linoleate
hydroperoxide .

59.  Singlet oxygen is capable of inducing damage
to the DNA .
 Lycopene shows greater quenching ability
than βcarotene (double ability )
 Comparing the structures opening of the β
ionine ring increases the quenching ability.

60.  Quenching ability of cartenoids not only
depends on triplet energy state that is the
length of the conjugated double bond system
but also on the functional groups .