Practical E Flashcards
Calculate P/O ratio steps
- Difference between State 1 + 2 / total oxygen saturation (y-axis) in length
- x1000
- conc. substance e.g. ADP/ P value
Effect of adding dinitrophenol + mechanism of action
- Normally ETC pumps protons out at same rate as ATP synthase allows them back in -> ATPS + ETC stops working when all ADP to ATP
- Dinitrophenol = alternative route into matrix allows ETC to pump protons out at same rate as dinitrophenol allows back in
- ATP synthase no longer has effect on ETC function -> so dp. on O2 conc. not dep. on ADP conc.
What happens if stirrer turned off
Stirrer ensures O2 evenly distributed -> Prevent O2 levels from being correctly measured
Calculate O2 consumption from ADP added
Amount of ADP/ ATP required to prod. 1 oxygen atom
-> /2 = O2
Oxygen consumption when amytal added to malate with + without dnp
O2 consumption stopped
Oxygen consumption when amytal added to succinate with + without dnp
No - O2 normal
Yes - Increased
Oxygen consumption when antimycin A added to malate with + without dnp
O2 consumption stopped
Oxygen consumption when antimycin A added to succinate with + without dnp
O2 consumption stopped
Oxygen consumption when oligomycin added to malate+ succinate with + without dnp
No- O2 consumption is slow, state 2 resp. continues
Yes - O2 consumption increased
Oxygen consumption when potassium cyanide added to malate + succinate with + without dnp
O2 consumption stopped
Oxygen consumption when rotenone added to malate with + without dnp
O2 consumption stopped
Oxygen consumption when rotenone added to succinate with + without dnp
No - O2 consumption normal
Yes - O2 consumption increase
Where do amytal and rotenone act?
O2 consumption doesn’t occur when malate is substrate so amytal prevents ETC at Complex 1
Where do antimycinA and potassium cynanide act
Inhibits ETC for both substrate => Complex 3/4 since ETC is inhibited even in presence of DNP (so no ATPS involvement
Oigomycin
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
An oxygen electrode measures
percentage oxygen saturation in reaction mixture
An oxygen electrode is calibrated against
a buffer saturated with oxygen to set 100% saturation + buffer depleted from oxygen by reaction with a strong reducing agent e.g. sodium dithionite
How are reaction chambers separated from electrodes in an oxygen electrode?
Teflon membrane
What reaction buffer is used in an oxygen electrode and what are the separate electrodes composed of?
KCl
Anode- Silver
Cathode - Platinum
Flavin and Ubiquinone are ______ nucleotides that can be involved in ________ + _________ transfer
- Nicotinamide
- Electron
- Hydrogen atom
In NAD or NADP, reactive group is
Nicotinamide ring
Reduction of oxidised co-enzymes involve __ electrons and result in formation of _____ and ______
2, NAD(P)+, associated proton
2 means by which flavin co-enzymes undergo redox reactions
Either a single 2 electron transfer or 2 single electron transfers via intermediate flavin free radical
Ubiquinone redox reaction involves
2 single electron transfers - most versatile
How does cytochrome undergo redox reactions?
Single electron transfer at iron atom
Why do redox potentials in cytochromes vary?
Different substituents in porphyrin ring
Protoporphyrin IX (haem)
Not covalently bound to histidine residues in the protein Hb, myoglobin, catalase and cytochromes b, b1 and P450
Haem C
Covalently bound to cysteine residues of the protein in cytochromes c + c1
Haem A
Anchored in membrane lipid by long hydrophobic tail in cytochromes a + a3
ETC electron carrier pathway
Flavoprotein -> Ubiquinone (1 ATP) -> Cytochrome b (1 ATP) -> c1 -> c -> Cytochrome oxidase (1 ATP + H2O)
Respiratory control index
Ratio between state 3 and state 4 -> indicates tightness of coupling between respiration and phosphorylation
Why is state 4 respiration usually faster than original rate?
some ATP is broken down by ATPase activities contaminating the preparation, and the resulting ADP is then re-phosphorylated by the intact mitochondria.
