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FREE RADICALES

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Title: FREE RADICALES


1
Free radicals
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
2
Free radicals
  • Definition
  • Free radical is a molecule or molecular
    fragment that contains one or more unpaired
    electrons in its outer orbits.

3
Electron configuration in free radial
4
Introduction
  • 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
Reactive oxygen species
  • 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
Endogenous generation of free radicles
  • Free radicles are generated in oxidative
    metabolism due to leak of electrons .
  • Flavoprotein linked oxidases like xanthine
    oxidase , L a 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
Endogenous generation of free radical
  • 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
contd
  • Free radicals are formed cytochrome P450
    reductase enzyme complex durinrg metabolism of
    xenobiotics .
  • ß oxidation of very long chain fatty acids in
    peroxisomes produces H2O2 .

9
Electron leak generates free radical
  • 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
contd
  • Cytochrome C oxidase does not release partially
    reduced intermediates , this crucial criterion
    meets by holding O2 tightly between Fe Cu atoms
    .

11
contd
  • 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 .

12
Electrons can be gained by univalent reduction
which may account for 1 -5 of total oxygen
consumption .
13
Free radicals from flavoprotien linked oxidases.
  • Flavoprotien linked oxidases
  • 1 ) Xanthine oxidase ,
  • 2) L a 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
contd
  • xanthine oxidase
  • Hypoxanthine xanthine
  • O2 O2
  • acetaldehyde dehydrogenase
  • Acetaldehyde
    acetate
  • O2 O2

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16
Respiratory burst
  • 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
contd
  • activation of inflammatory cell
  • drastic increase in consumption of oxygen
    (respiratory burst )
  • 10 of oxygen uptake by macrophage is used for
    free radical generation .

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19
contd
  • 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
contd
  • 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
Free radicals from metabolism of eicosanoids
  • 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
Free iron producing free radicals
  • Super oxide ion can release iron from ferritin .

23
contd
  • 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.

24
Cytochrome P 450 reductase
25
Other factors
  • 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
Lipid peroxidation
  • 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
Initiation phase
  • 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
Propagation phase
  • 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
Termination phase
  • The above reactions would proceed unchecked till
    a peroxyl radical reacts with another peroxyl
    radical to form inactive products .
  • ROO ROO RO- -ORO2
  • R R R - - R
  • ROO R RO- -OR

30
Intracellular antioxidants
  • Super oxide dismutase
  • Catalase
  • Glutathione peroxidase
  • Cytochrome oxidase

31
Super oxide dismutase
  • 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 .

32
Super oxide dismutase
33
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34
contd
  • 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 .

35
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36
Catalase
  • 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
contd
  • 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
Contd
  • 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
contd
  • 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
Contd
  • 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 2H2oO2

41
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42
Glutathione peroxidase
  • 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 .

43
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46
Membrane antioxidants
  • Vitamin E lipid soluble , chain breaking
    antioxidant.
  • ßcarotene 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
Extra cellular antioxidants
  • 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
    stoichiometric O2 scavenging ,binds copper ions
    utilizes H2O2 for reoxidation of copper .

48
contd
  • 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
Chain breaking antioxidants
  • Water soluble urate , ascorbates ,
  • thiols ,bilirubin,
  • flavanoids.
  • Lipid soluble tocopherol ,
  • ubiquinol 10 ,
  • ß carotene .
  • Urate vitamin E acts in lipid phase to trap
    ROO radicals .

50
Preventive antioxidants
  • 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
  • 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 atocoferol vitamin appears to concentrate
    at these sites.

52
contd
  • 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 vitamin C
    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
contd
  • ROO TocOH ROOH TocO
  • ROO TocO ROOH non free

  • radical product

54
contd
  • ROO TocOH ROOH TocO
  • ROO TocO ROOH non free

  • radical product

55
ss
56
contd
  • The antioxidant action of vitamin E is effective
    at high oxygen concentrations , it is
    concentrated in lipid structures exposed to high
    O2 partial pressures such as the erythrocyte
    membrane , membranes of respiratory tree the
    retina.

57
contd
  • 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
Vitamin A as antioxidant
  • Carotenoids are capble of quenching singlet
    molecular oxygen .
  • Carotenoids like lycopene ,ß carotene , are
    important biological molecules that can
    inactivate electronically excited molecules by
    process called quenching.
  • ßcarotene related compounds can acts as chain
    breaking antioxidant.
  • Can acta as preventive antioxidant by decreasing
    the formation of methyl linoleate hydroperoxide .

59
contd
  • Singlet oxygen is capable of inducing damage to
    the DNA .
  • Lycopene shows greater quenching ability than
    ßcarotene (double ability )
  • Comparing the structures opening of the ß
    ionine ring increases the quenching ability.

60
contd
  • 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 .

61
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