FREE RADICLES

1. Free radicals M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

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

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 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. 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 . • β 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 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. contd xanthine oxidase Hypoxanthine xanthine O2 O2 · acetaldehyde dehydrogenase Acetaldehyde acetate O2 O2 ·

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 .

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- -OR+O2 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

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 .

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 2H2o+O2

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 .

46. Membrane antioxidants • 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. 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 , β 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 .