l10

Question 1

give three examples of reactive oxygen species

Answer 1

H2O2, superoxide anion 02-, hydroxyl radicals OH

Question 2

What are the cellular sources of reactive oxygen species (ROS)

Answer 2

ETC in mitochondria and NAPDH oxidase complex by phagocytes

Question 3

What is ixidative stress?

Answer 3

ROS level > antioxident capacity of the cell

Question 4

What can be oxidatively damaged by ROS?

Answer 4

proteins, lipids and DNA

Question 5

What are the things that occur as a result of o2 damage of proteins liids and DNA?

Answer 5

apoptises, cell death, cancer, ND, diabetes, agening

Question 6

What is the free radical of aging?

Answer 6

aging occurs as a result of accummulation of oxidantion

Question 7

What are the endogenous sources of O2 in cells?

Answer 7

mitochondria, peroxisomes, NAPH oxidase

Question 8

What are the exogenous sources of ROS?

Answer 8

UV, chemotherapy, env. tociins and ionising raidation

Question 8

What are the antioxidant defences

Answer 8

Enzymatic (CAT SOD) and non-enxymatic (gluytathionine)

Question 8

What are the two sonesquences from ROS that could result in againg

Answer 8

impaired physiological fn and accumulation of O2 damage

Question 8

Are all ROS harmful?

Answer 8

not necesaruly

Question 8

What sort of things can protein oxidation signal for in cells?

Answer 8

Growth, division, migration, differentiatio

Question 8

what’s the key amino acid when it comes to protein oxidation

Answer 8

cysteine

Question 8

What can the oxidation of thiols regulate?

Answer 8

protein function, activity and cellular localisation

Question 8

What is the side chain in cysteine?

Answer 8

thiol (SH)

Question 9

Does sulphenic acid make disulphide bonds? If yes, why, and if so is it the only thing it forms

Answer 9

Yes. Sulphenic acid very reactive. No. Forms two types of disulphide bonds (intra and inter) and forms sulphenamide (S-NH)

Question 9

What is the product of thiol being exposed to low levels of H2O2?

Answer 9

sulphenic acid (one thiol (SH), one S-OH)

Question 9

What happens when sulphenic acd is further oxidised

Answer 9

Turns into sulphinic acid (S=O,OH)

Question 9

What can the ocidation of sulphenic acid be reversed by? What type of molecule is this?

Answer 9

thrioredoxin, Trx. Protein

Question 10

Is sulphenic to sulphinic acid reversible

Answer 10

yes, srx.

Question 11

What determines the specificity of protein oxidation

Answer 11

uncommon acid, not as accessible, and only a few are redox sensitive.

Question 12

Why are only few cystine residues redox sensitive

Answer 12

depends on pKa

Question 13

What is pKa?

Answer 13

pH at which 50% of pop. de-protonated

Question 14

What is the common pKa value of most protein thiols?

Answer 14

8

Question 15

What is the pH of cells?

Answer 15

7

Question 16

What is the redox sensitive thiols pKa?

Answer 16

3-5.5

Question 17

How do we get redox sensitive thiol groups?

Answer 17

neighbouring AA - needs to be close to proton accepting AA, His, lys, Arg.

Question 18

How does thioredoxin reduce oxidised cystine residues?

Answer 18

using electrons from NADPH

Question 19

How is oxidised thioredoxin reduced?

Answer 19

thioredoxin reductase

Question 20

How does thioredoxin reductase reduce oxidised thioredoxin?

Answer 20

electrons from NADPH

Question 21

How does srx12 reduce cysteine-sulphinic acid

Answer 21

ATP - dep

Question 22

What is the issue with oxidation as a PTM?

Answer 22

unstable as breaking open cells = artificial o2.

Question 23

What is the solution to the artificial oxidation of proteins?

Answer 23

acid lysis, lowers pH of the cell so cystiene residues remain protonated. and cysteine0binding chem. only bind to reduced custeine residues

Question 24

How are intermolecular disulphides detected?

Answer 24

DTT - reducing agent, reduces disulphide bonds

Question 25

oxidation detected by an increase in _____ and is ____ sensitive

Answer 25

size, DTT

Question 26

What is the issue w western blotting and DTT?

Answer 26

what's the interacting partner & we need specific Ab

Question 27

What are cystine binding chemicals

Answer 27

only bind to reduced cysteine residues

Question 28

what are the three cystine binding chemicals and MW

Answer 28

NEM - 0.1 kDa block cyst. residues AMS, 0.5 kDa PEG-Mal 2-5 kDa - protein interaction ?

Question 29

why are proteins from cells extracted under acidic conditions

Answer 29

avoid artificial oxidation

Question 30

How is disulphide interactions noted?h

Answer 30

No AMS will bind to the disulphide bond

Question 31

How does AMS binding affect mobility on SDS page?

Answer 31

heavier MW, slower

Question 32

What are the advantages of using SDS page

Answer 32

visualise protein oxidation by mobility change

Question 33

What are the issues in using SDS PAGE?

Answer 33

Type of oxidation is not revealed and again specific antibodies are needed

Question 34

What do we use to detect intramolecular disulphide

Answer 34

PEG-Mal

Question 35

How do we detect intraM disulp. bonds?

Answer 35

Block SH groups with NEM, reduce using DTT, and then label using PEG-Mal.

Question 37

What's the issue with disulph. bond detection

Answer 37

is the cysteine res. modified? need specific Ab/