l10 Flashcards
give three examples of reactive oxygen species
H2O2, superoxide anion 02-, hydroxyl radicals OH
What are the cellular sources of reactive oxygen species (ROS)
ETC in mitochondria and NAPDH oxidase complex by phagocytes
What is ixidative stress?
ROS level > antioxident capacity of the cell
What can be oxidatively damaged by ROS?
proteins, lipids and DNA
What are the things that occur as a result of o2 damage of proteins liids and DNA?
apoptises, cell death, cancer, ND, diabetes, agening
What is the free radical of aging?
aging occurs as a result of accummulation of oxidantion
What are the endogenous sources of O2 in cells?
mitochondria, peroxisomes, NAPH oxidase
What are the exogenous sources of ROS?
UV, chemotherapy, env. tociins and ionising raidation
What are the antioxidant defences
Enzymatic (CAT SOD) and non-enxymatic (gluytathionine)
What are the two sonesquences from ROS that could result in againg
impaired physiological fn and accumulation of O2 damage
Are all ROS harmful?
not necesaruly
What sort of things can protein oxidation signal for in cells?
Growth, division, migration, differentiatio
what’s the key amino acid when it comes to protein oxidation
cysteine
What can the oxidation of thiols regulate?
protein function, activity and cellular localisation
What is the side chain in cysteine?
thiol (SH)
Does sulphenic acid make disulphide bonds? If yes, why, and if so is it the only thing it forms
Yes. Sulphenic acid very reactive. No. Forms two types of disulphide bonds (intra and inter) and forms sulphenamide (S-NH)
What is the product of thiol being exposed to low levels of H2O2?
sulphenic acid (one thiol (SH), one S-OH)
What happens when sulphenic acd is further oxidised
Turns into sulphinic acid (S=O,OH)
What can the ocidation of sulphenic acid be reversed by? What type of molecule is this?
thrioredoxin, Trx. Protein
Is sulphenic to sulphinic acid reversible
yes, srx.
What determines the specificity of protein oxidation
uncommon acid, not as accessible, and only a few are redox sensitive.
Why are only few cystine residues redox sensitive
depends on pKa
What is pKa?
pH at which 50% of pop. de-protonated
What is the common pKa value of most protein thiols?
8
What is the pH of cells?
7
What is the redox sensitive thiols pKa?
3-5.5
How do we get redox sensitive thiol groups?
neighbouring AA - needs to be close to proton accepting AA, His, lys, Arg.
How does thioredoxin reduce oxidised cystine residues?
using electrons from NADPH
How is oxidised thioredoxin reduced?
thioredoxin reductase
How does thioredoxin reductase reduce oxidised thioredoxin?
electrons from NADPH
How does srx12 reduce cysteine-sulphinic acid
ATP - dep
What is the issue with oxidation as a PTM?
unstable as breaking open cells = artificial o2.
What is the solution to the artificial oxidation of proteins?
acid lysis, lowers pH of the cell so cystiene residues remain protonated. and cysteine0binding chem. only bind to reduced custeine residues
How are intermolecular disulphides detected?
DTT - reducing agent, reduces disulphide bonds
oxidation detected by an increase in _____ and is ____ sensitive
size, DTT
What is the issue w western blotting and DTT?
what’s the interacting partner & we need specific Ab
What are cystine binding chemicals
only bind to reduced cysteine residues
what are the three cystine binding chemicals and MW
NEM - 0.1 kDa block cyst. residues
AMS, 0.5 kDa
PEG-Mal 2-5 kDa
- protein interaction ?
why are proteins from cells extracted under acidic conditions
avoid artificial oxidation
How is disulphide interactions noted?h
No AMS will bind to the disulphide bond
How does AMS binding affect mobility on SDS page?
heavier MW, slower
What are the advantages of using SDS page
visualise protein oxidation by mobility change
What are the issues in using SDS PAGE?
Type of oxidation is not revealed and again specific antibodies are needed
What do we use to detect intramolecular disulphide
PEG-Mal
How do we detect intraM disulp. bonds?
Block SH groups with NEM, reduce using DTT, and then label using PEG-Mal.
What’s the issue with disulph. bond detection
is the cysteine res. modified? need specific Ab/
