Practical E

Question 1

Calculate P/O ratio steps

Answer 1

1. Difference between State 1 + 2 / total oxygen saturation (y-axis) in length
2. x1000
3. conc. substance e.g. ADP/ P value

Question 2

Effect of adding dinitrophenol + mechanism of action

Answer 2

1. Normally ETC pumps protons out at same rate as ATP synthase allows them back in -> ATPS + ETC stops working when all ADP to ATP
2. Dinitrophenol = alternative route into matrix allows ETC to pump protons out at same rate as dinitrophenol allows back in
3. ATP synthase no longer has effect on ETC function -> so dp. on O2 conc. not dep. on ADP conc.

Question 3

What happens if stirrer turned off

Answer 3

Stirrer ensures O2 evenly distributed -> Prevent O2 levels from being correctly measured

Question 4

Calculate O2 consumption from ADP added

Answer 4

Amount of ADP/ ATP required to prod. 1 oxygen atom

-> /2 = O2

Question 5

Oxygen consumption when amytal added to malate with + without dnp

Answer 5

O2 consumption stopped

Question 6

Oxygen consumption when amytal added to succinate with + without dnp

Answer 6

No - O2 normal

Yes - Increased

Question 7

Oxygen consumption when antimycin A added to malate with + without dnp

Answer 7

O2 consumption stopped

Question 8

Oxygen consumption when antimycin A added to succinate with + without dnp

Answer 8

O2 consumption stopped

Question 9

Oxygen consumption when oligomycin added to malate+ succinate with + without dnp

Answer 9

No- O2 consumption is slow, state 2 resp. continues

Yes - O2 consumption increased

Question 10

Oxygen consumption when potassium cyanide added to malate + succinate with + without dnp

Answer 10

O2 consumption stopped

Question 11

Oxygen consumption when rotenone added to malate with + without dnp

Answer 11

O2 consumption stopped

Question 12

Oxygen consumption when rotenone added to succinate with + without dnp

Answer 12

No - O2 consumption normal

Yes - O2 consumption increase

Question 13

Where do amytal and rotenone act?

Answer 13

O2 consumption doesn’t occur when malate is substrate so amytal prevents ETC at Complex 1

Question 14

Where do antimycinA and potassium cynanide act

Answer 14

Inhibits ETC for both substrate => Complex 3/4 since ETC is inhibited even in presence of DNP (so no ATPS involvement

Question 15

Oigomycin

Answer 15

State 2/3 respiration always observed -> all components of ETC are working at relatively high rate -> Dnp restores re-entry of photons -> inhibit ATP synthase

Question 16

An oxygen electrode measures

Answer 16

percentage oxygen saturation in reaction mixture

Question 17

An oxygen electrode is calibrated against

Answer 17

a buffer saturated with oxygen to set 100% saturation + buffer depleted from oxygen by reaction with a strong reducing agent e.g. sodium dithionite

Question 18

How are reaction chambers separated from electrodes in an oxygen electrode?

Answer 18

Teflon membrane

Question 19

What reaction buffer is used in an oxygen electrode and what are the separate electrodes composed of?

Answer 19

KCl
Anode- Silver
Cathode - Platinum

Question 20

Flavin and Ubiquinone are ______ nucleotides that can be involved in ________ + _________ transfer

Answer 20

1. Nicotinamide
2. Electron
3. Hydrogen atom

Question 21

In NAD or NADP, reactive group is

Answer 21

Nicotinamide ring

Question 22

Reduction of oxidised co-enzymes involve __ electrons and result in formation of _____ and ______

Answer 22

2, NAD(P)+, associated proton

Question 23

2 means by which flavin co-enzymes undergo redox reactions

Answer 23

Either a single 2 electron transfer or 2 single electron transfers via intermediate flavin free radical

Question 24

Ubiquinone redox reaction involves

Answer 24

2 single electron transfers - most versatile

Question 25

How does cytochrome undergo redox reactions?

Answer 25

Single electron transfer at iron atom

Question 26

Why do redox potentials in cytochromes vary?

Answer 26

Different substituents in porphyrin ring

Question 27

Protoporphyrin IX (haem)

Answer 27

Not covalently bound to histidine residues in the protein Hb, myoglobin, catalase and cytochromes b, b1 and P450

Question 28

Haem C

Answer 28

Covalently bound to cysteine residues of the protein in cytochromes c + c1

Question 29

Haem A

Answer 29

Anchored in membrane lipid by long hydrophobic tail in cytochromes a + a3

Question 30

ETC electron carrier pathway

Answer 30

Flavoprotein -> Ubiquinone (1 ATP) -> Cytochrome b (1 ATP) -> c1 -> c -> Cytochrome oxidase (1 ATP + H2O)

Question 31

Respiratory control index

Answer 31

Ratio between state 3 and state 4 -> indicates tightness of coupling between respiration and phosphorylation

Question 32

Why is state 4 respiration usually faster than original rate?

Answer 32

some ATP is broken down by ATPase activities contaminating the preparation, and the resulting ADP is then re-phosphorylated by the intact mitochondria.