L22 Oxidative stress pt 2 Flashcards
When does hydrogen peroxide become an issue?
When iron ions are free in the cell/tissue. Fenton reaction.
Promotes formation of the hydroxyl radical.
Issue in certain regions of the brain.
What is a key defense against free radicals?
Superoxide dismutase (SOD).
Catalyses formation of superoxide into hydrogen peroxide.
Three forms found in humans
What is the role of the glutathioen system?
A key antioxidant pathway in the cell.
Good at removing hydrogen peroxide.
How does glutathione remove ROS?
Reduced glutathione reacts with hydrogen peroxide.
Is oxidised by this. This removes hydrogen peroxide.
NADP dependent.
What residues of glutathione does hydrogen peroxide oxidise?
Cysteine residues. Forms disulphide bridges.
H2O2 targets cysteine containing proteins
What are the peroxidoxins?
Proteins present in the cell, deactivate hydrogen peroxide.
Their cysteine resides are oxidised by h2o2.
How can ROS be measured?
Visual/quantifiable plate reading fluorescence assays.
Dyes directly can be used with colorimetric change when themselves are directly oxidised.
CM-DCFH-DA. DCFH.
What is the disadvantage to using dyes such as DCFH?
Non-specific, reacts with many ROS, not just h2o2, superoxide etc.
A broad assesser of oxidative stress.
What dye can be used for directly measuring h2o2
Ample red.
Reacts with h2o2 in a 1:1 reaction, catalysed by HRP, generates highly fluroescent product, resofurin.
HRP catalyses reaction of h2o2 to hydrogyl radical OH, which then oxidises amplex red to give colour change.
What state of h2o2 is amplex red used to measure?
Secreted h2o2, not intracellular.
What is a more in-depth genetic approach to studying h2o2
roGFP-tagging.
Redox-sensitive GFP, sensitive to oxidation by addition of cysteine residues.
Means GFP is ‘inactivated’ when oxidised and does not fluoresce.
What is a good way of measuing superoxide?
Using MitoSOX dye.
Red mitochondrial superoxide indicator.
Accumulates within mitochondria, readily oxidied by superoxide.
Why is superoxide difficult to measure?
How is this overcome?
It is short lived.
Meausre the enzyme, SOD.
Measures the condition of SOD after superoxide production.
What is the best way to look at total antioxidant capacy in the cell?
Measuring glutathione.
Total glutathione in cell can be measured easily using ellman’s reagent. Reacts with glutathione, produces coloured product, quantifiable with platereader.
What is a more sophisticated method of measuring glutahtione content?
Fluorescent kit.
Luciferin probe reacts only to reduced glutathione.
All glutathione is reduced to measure total, and oxidation of glutathione is blocked.
What is the comet assay?
Assay to measure DNA damage / genotoxicity.
Single cell gel electrophoresis (SCGE)
How is the comet assay used for measuring oxidative level of cells?
ROS breaks phosphodiester backbone of DNA, so higher ROS, larger ‘tail’ in comet assay.
How can lipid peroxidation be used?
Indicator of free radical formation, key indicator of oxidative stress.
Hydroxyl adicals reacts near to c=c in unsaturared FAs.
What is malondialdehyde (MDA)
Product of lipid peroxidation, measured to quantify this, using TBARS assay.
What is a better assay for measuring lipid peroxidation than TBARS?
Lipid peroxidation assay kit. Using fluorescence.
What PTMs occur as a product of ROS?
Oxidative/Nitrosative mod.
Phospho, ubi, acetyl, as a consequence of ROS
What is the Keafp1/Nrf2 signalling pathway?
Pathway that regulates oxidative homeostasis globally.
How is Nrf2 kept in normal conditions?
Kept inactive, bound to Keap1.
This is tagged by ubi for degradation.
It is constantly made, and constantly degradated due to Cul 3.
Under oxidative stress, Keap1 is oxidised and sensitive due to cysteine residues. This means Nrf2 is released from Keap1.
Means Nrf2 is active, translocates to nucleus and is a TF, binds to antioxidant response element genes.
