Oxidative Stres pt. 2 Flashcards
Reaction of oxygen free radicals
the first reaction of a hydroxyl radical is to remove a hydrogen atom from any molecules in the cells to generate water + alkyl radical the alkyl radical can then react with oxygen to form a peroxy radical
hydroxyl radical is
highly reactive + short lived
Mechanism of lipid peroxidation
- attack on a species that can abstract a H from a methylene carbon on the side chain 2. H atom is now a free radical & it leaves behind a carbon center radical 3. in aerobic cells it undergoes molecular rearrangement 4. then reacts with O2 to generate a peroxy radical
peroxy radicals can attack
membrane proteins; they are capable of abstracting hydrogen from adjacent fatty acid side chains in a membrane therefore PROPAGATING the chain reaction of lipid peroxidation
length of lipid peroxidation propagation chain via peroxyl radicals is dependent on many factors
- lipid/protein ratio in a membrane 2. fatty acid composition 3. oxygen concentration 4. presence within the membrane of chain-breaking antioxidants like AH which interrupt the chain reaction by providing easily-donatable hydrogen
if two radicals react
both radicals are eliminated
if a radical reacts with a nonradical
another radical will be produced
which reactive species of oxygen initiate lipid peroxidation?
most involve transition metal ions the oxidized forms of these transition metal ions accelerate peroxidation if a reducing agent is present
hydroxyl radical is almost always detectable in metal ion-dependent peroxidation systems; addition of catalase rarely inhibits the peroxidation observed
this indicates that the metal ions can react with lipid hydroperoxides present in the membrane preparation to produce lipid alkoxyl radicals and lipid radicals - both are capable of initiating the chain reaction of lipid peroxidation
biological consequences of lipid peroxidation
- impairment of membrane function 2. decreased fluidity 3. inactivation of membrane-bound receptor and enzymes 4. increased non-specific permeability to ions such as calcium **lipid radicals and lipid alkoxyl radicals(which are products of decomposition of lipid hydroperoxides) can abstract further hydrogen atoms from fatty acid chains**
major aldehyde produced by lipid peroxidation is 4-hydroxynonenal (HNE), HNE is
cytotoxic in a dose-dependent manner to a broad spectrum of cells implicated in numerous forms of cell injury stimulates neutrophil chemotaxis modulate adenylate and guanylate cyclase activity stimulate phospholipases it’s genotoxic + carcinogenic + cause cell death
another aldehyde generated by lipid peroxidation is malondialdehyde
can cause cross-linking, polymerization of membrane proteins alter intrinsic membrane properties like deformability, ion transport, enzyme activity, aggregation of cell surface determinants it’s diffusable - it can interact with nitrogenous bases of DNA it’s mutagenic + genotoxic + carcinogenic
biological effects of aldehydes: 100micromolar
nonspecific cytotoxic effects leading to rapid cell death rapid depletion of glutathione, decrease in protein thiols, onset of lipid peroxidation, disturbance of Ca homeostasis, inhibition of DNA, RNA and protein synthesis, inhibition of respiration and glycolysis, lactate release (it’s unlikely it would actually reach this concentration)
biological effects of aldehydes: 1-20micromolar
inhibit DNA and protein synthesis stimulate phospholipase A2 inhibit oncogene c-myc expression activate heat shock and heme oxygenase-1 gene expression **may all occur following oxidative stress**
biological effects of aldehydes: <0.1micromolar
likely to occur at basal physiological level stimulation of chemotaxis modulation of adenylate cyclase activity weak stimulation of guanylate cyclase stimulation of phospholipase C
Denaturation of proteins by aldehydes
aldehydes are long lived, so they diffuse
Denaturation of proteins by aldehydes: HNE has 3 main functional groups
- aldehyde group 2. CC double bond 3. hydroxyl group
Denaturation of proteins by aldehydes: HNE readily is reacted with glutathione to form
a saturated aldehyde, this undergoes an intramolecular rearrangement to the 5-membered cyclic hemiacetal **the aldehyde groups of HNE are preserved in all conjugated amino acids - may undergo secondary reactions with the same amino groups of lysine residues of the same/different protein to yield intra- and inter-subunit cross-links via schiff base formation **
the level of proteins that are conjugated with HNE is increased following
oxidative stress - representing a consequence of the increased lipid peroxidation
Denaturation of proteins by aldehydes: malondialdehyde (MDA)
major aldehyde released by peroxidized lipids, known to react with the e-amino group of lysine residue in a protein to form unstable amino propenal & intra-/inter- protein imine adducts - this contributes to intra- or inter- protein cross-links
Denaturation of proteins by aldehydes: MDA modified proteins induce
induce inflammatory responses and are recognized by the innate immune system found in atherosclerosis, age-related macular degeneration, other chronic degeneratie diseases
clinical relevance of lipid peroxidation: age-related macular degeneration
medical condition which affects older people; results in loss of vision in the center of the visual field because of damage to the retina may partly result from many environmental and genetic factors such as tissue inflammation and oxidative stress due to cigarette smoking /light exposure
Protein oxidation
generated by radiolysis and metal-catalyzed reactions
oxidation of free amino acids residues in proteins by ionizing radiation
effects of ionizing radiation on amino acids and proteins in oxygenated solutions are mainly attributable to the formation of hydroxyl radial, peroxide radical and hydroperoxyl radical
alphatic amino acids
leads to the formation of NH4+ and alpha ketoacids
depletion of oxygen in reaction mixtures during radiolysis
may suppress the generation of peroxyl radical and favor reductive deamination as well as variety of carbon-centered radical combination interaction to form mono- & di-amino dicarboxylic acid derivatives
aromatic amino acids
indole ring of tryptophan and aromatic group of tyrosine and phenylalanine are PRIMARY targets of oxygen radicals produced during radiolysis , alpha-hydrogen abstraction and deamination are relatively minor events
Major product of tryptophan oxidation is
formylkynurenine
oxidation of tyrosine forms
3,4-dihyroxyphenylalanine
oxidation of phenylalanine leads to the production of
tyrosine and other hydroxy derivatives
proteins in the absence of oxygen
there is little or no fragmentation of the protein but considerable amounts of high-molecular-weight aggregates are formed
proteins in the presence of oxygen
little or no aggregation occurs but there is considerable fragmentation
oxygen-mediated cleave of the polypeptide chain
occurs by the alpha-amidation pathway
alpha hydrogen of amino acid residues (and methyl, methylene groups) in proteins are major sites of
hydroxyl attack
proteins are mostly converted to what when exposed to radiolysis in the absence of oxygen?
converted to high-molecular weight aggregates - these aggregates are known to result from formation of inter-protein cross-linking reactions and from hydrophobic and electrostatic interactions
metal-catalyzed oxidation(MCO) of amino acid residues in proteins: free amino acids
in the presence of Fe(III) or Cu(II), oxygen and an electron donor MCO systems are able to oxidize free amino acids and amino acid residues in proteins
the MCO system with free amino acids includes
nonenzymatic systems and enzymatic systems (including NADPH dehydrogenase, xanthine oxidase, CYP450 reductases, quinone reductase)
metal-catalyzed oxidation of amino acid residues in proteins: proteins
more than 40 proteins have been shown to be sensitive to metal-catalyzed reaction: His, Arg, Lys, Pro, Met and Cys are the most common
metal-catalyzed oxidation of amino acid residues in proteins: only one or only a few amino acid residues in a given protein are modified by the MCO systems but how many amino acids are?
all amino acids are susceptiable to oxidation by free radicals generated during radiolysis
carbonyl group
a functional group composed of a carbon atom double-bonded to an oxygen atom
superoxide anion radical can act either as an
univalent oxidant or reductant
superoxide anion radical - family of dehydrogenases contains clusters at their active sites, the group includes
alpha, beta-dihydroxyacid dehydratase, 6-phosphogluconate dehydrogenase and acontiase they undergo rapid oxidation by superoxide radical with a resultatnt loss of Fe(II) fromt eh cluster and concomitnat inactivation - inactivation affacts various pathways in cellular metabolism (like branched-chain amino acid synthesis, pentose phosphate pathway and tricarboxylic acid cycle)
release of Fe(II) from the cluster sets the stage for the Fenton reaction
where a hydroxyl radical is produced this reaction is the basis for cooperation between superoxide and hydrogen peroxide to attack a variety of cellular targets also predicts the protective role of SOD in cells against hydrogen peroxide induced damage as the increas SOD activity will prevent the inactivation of cluster and release of Fe(II) to participate in the Fenton reaction
Molecular mechanism of NO reactivity
NO reacts with oxygen, superoxide and transition metals, each of the products of these reaction can participate in further reactions with the thiol groups
NO’s Interactions with metals
react directly with other radicals or one electron donors/acceptors such as the iron in heme
NO can also inhibt the activity of enzymes
utilize the heme prosthetic group in catalysis - this is well exaple in the inhibition of cytochrome P450 enzymes
the binding of NO to either Fe(II) to Fe(III) heme
attenuates oxygenase activity
binding between the NO and heme iron induces
a conformational change that displaces iron out of the plane of the porphyrin ring and acitvates the enzyme guanylate cyclase catalyzes the conversion of guanosine-5-triphosphate to cyclic guanosine 3,5monophosphate results in relaxation of smooth muscle(vasodialation) -this mechanism may be responsible for the therapeutic effects of nitroso-vasodialtors
mechanism for interaction between NO and the thiol groups of cysteinsin proteins
is not understood reaction probably involves formation of NO+ through oxidation of NO by transition metals or reaction of NO with O2
Genotoxicity of NO: exposure to NO has been shown to cause
deamination of DNA & RNA
peroxynitrite, formed by reaction between NO and superoxide radical
believed to play a major role in radical-induced cell damage since it’s a stable molecule for several weeks
peroxynitrite can nitrate phenolic rings such as tyrosine residues in proteins
tyrosine phosphorylation, altered protein function, increased protein turnover due to enhanced proteolysis, initiation of autoimmune reactions to antigenic haptens
peroxynitrite but not NO
can rapidly inactivate aconitase its activity can then be totally restored by incubation with tiols and ferrous ion indicating that oxidation cluster by peroxynitrite may play a role in enzyme inactivation
peroxynitrite can either react with the sulfhydryl groups of proteins or become protonated ot form peroxynitrous acid which decays rapidly and gives rise to
hydroxyl radical and ntirogen dioxide these radicals will then cause protein oxidation and lipid peroxidation
