Regulation of Proteins: Haemoglobin/Myoglobin/Clotting cascade Flashcards Preview

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Flashcards in Regulation of Proteins: Haemoglobin/Myoglobin/Clotting cascade Deck (50)
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1
Q

What kind of binding causes the sigmoidal curve of Hb and O2

A

Cooperative - with each O2 bound - Hb increases affinity for O2

2
Q

Where does oxygen bind in Hb and Myoglobin?

A

To haem groups - Fe can bond 2 Oxygen molecules (O2) either side of the plane of the pyrrole ring

3
Q

What happens to the structure of Haem in myoglobin on binding O2. Is it that relevant compared to the change in structure of Hb on binding O2?

A

Fe normally sits just below the ring, on O2 binding Fe moves up into plane of ring - movements causes movement of HisF8a nd small change in overall protein conformation. Not really as relevant as the change in Hb

4
Q

What is p50

A

Partial pressure giving 50% oxygen saturation

5
Q

Which has higher affinity for O2 myoglobin or Hb

A

Myoglobin

6
Q

What effect does cooperativity of O2 binding in Hb have on % of O2 delivery to tissues?

A

With cooperativity there is a 66% difference between the lungs and tissue meaning 66% O2 can be offloaded at tissues. Without cooperativity this would only be 38%

7
Q

What would be the consequence a the lungs/tissues of something that has very high affinity for O2

A

Good uptake at lungs low offloading at tissues

8
Q

How many BPG bind per Hb, what effect does it have? What effect on the oxyhaem cure? When would you see an increase in BPG?

A

1 per Hb
Reduces affinity for O2
At high altitude - increased O2 release at tissues.
Shifts curve to right

9
Q

Whats the conc of BPG at RBCs?

A

5mM

10
Q

What is the BOHR effect? What does it ensure? What does the BOHR effect do to the oxyhaem graph?

A

H+ and CO2 both bind Hb molecules and lower affinity for O2. Ensures release of at metabolically active tissues. Shifts to right

11
Q

What effect does CO have on Hb and humans? How do you overcome? Is it more or less dangerous in anaemic patients at 50% CO?

A

CO binds to Hb with higher affinity that O2 - prevents O2 uptake. Have to increase PPO2 to overcome. E.g. CO poisoning treatment in hosp is O2. Think less? but not sure why - something about lower affinity maybe so more O2 release at tissues so will survive longer?

12
Q

What happens to HbF with alpha-2 gamma-2 chains after birth?

A

Subsides - is foetal Hb

13
Q

What is alpha vs beta thalassemia?

A

Decreased or absent alpha or beta chains

14
Q

What happens in the different types of Thalassemia? Do symptoms for each appear before or after birth?

A

alpha - before birth
beta- baby - birth onwards

2 Beta alleles Loss of 1 beta chain = mild, loss of 2 = severe. Alpha chains precipitate on their own - severe anaemia

Hb Bart worst - usually stillborn

4 Alpha alleles Loss of 1 is silent, loss of any 2 = range of mild symptoms, loss of 3 severe, loss of all 4 = Hydrops fatalis - death

15
Q

Are alpha or beta chains unable to form stable tetramers on their own?

A

alpha are unable to form stable tetramers

beta chains can form stable tetramers with increased affinity for O2

16
Q

What are the symptoms of Thalassemia and what is the treatment?

A
Lassitude - weariness/tired
Swollen spleen
Yellow Sclera 
Increased reticulocytes in response.
Treatment is regular blood transfusion
17
Q

What is the basic structure of Hb?

A

2 alpha 2 beta chains in a tetramer

4 haem groups each carry oxygen

18
Q

What are the two Hb states? How are they formed?

A

R - relaxed high affinity = due to small rotation of subunits by 15 degrees exposes haem groups

T- tense low affinity = haem not exposed

19
Q

Which state of Hb does O2 binding promote stabilisation of?

A

R

20
Q

6 ways in which enzyme activity can be regulated in the short term?

A
Proteolytic cleavage
Covalent modification
Allosteric effectors
Isoenzymes
Substrate amount
Product inhibition
21
Q

What is product inhibition? Give an example of regulation

A

Where product inhibits forward reaction e.g. with Glycogen breakdown signals also inhibit synthesis

22
Q

What are isoenzymes and give an example of regulation

A

Same enzyme different catalytic properties - e.g. Gluco and Hexokinase

23
Q

What are allosteric effectors and give an example of regulation.

A

Regulation via a site that isn’t the active site can be activating or inhibiting. E.g. PFK - ATP is an allosteric inhibitor.

24
Q

Do allosterically regulated enzymes show Michaelis Mentes kinetics?

A

No- show sigmoid not hyperbolic. Sigmoid is between high affinity R state and low affinity T state

25
Q

What are zymogens? Give an example of regulation

A

Precursor proteins that are activated when needed via proteolytic cleavage. e.g. Digestive enzymes, blood clotting cascade.

26
Q

What is covalent modification as an enzyme regulation method? Give an example

A

Covalently modifying protein either reversibly or irreversibly. E.g. Phosphorylation reversible.

27
Q

How do you phosphorylate something? Why is it so useful (5)?

A

Add terminal P of ATP to ser, thr, tyr OH group.

1) Links energy status of cell to metabolism through ATP
2) Reversible
3) Amplification
4) Can then make H+ bonds with effector molecule
5) Adds two - charges can completely change protein shape

28
Q

Which are two long term methods of enzyme regulation to increase activity?

A

1) Increased synthesis

2) Reduced degradation - ubiquitin pathway stimulates degradation

29
Q

What enzyme regulatory mechanisms control the blood clotting cascade and how ?

activating
inhibiting
clot breakdown

A

Zymogens - many of the factors are proteases - cleave next bit.

Amplification - cascade and + feedback

Allosteric activation - e.g. Thrombin on factor V, VIII and XI

Degradation by proteases - factor V and VIII degraded by protein C

Specific inhibitors - Bind to Thrombin (AT3) and block its activity. Enhanced by heparin. AT3 does not bind to thombomodulin bound thrombin.

Fibrinolysis by proteolytic cleavage of zymogen plasminogen to plasmin which breaks clot - used in brain clots

30
Q

What do endopeptidases do in the clotting cascade? What does this lead to?

A

Cleave zymogens to activate - many are Factors. Leads to amplification of signal, each factor activates the next.

31
Q

Is Fibrin an enzyme?

A

No

32
Q

Is Thrombin an enzyme?

A

Yes

33
Q

What is the role of Gla domains Kringle domains and calcium in the coagulation pathway?

A

Gla domains target the protease function of prothrombin to the appropriate site for activation (site of damage) . Kringle domains keep prothrombin in inactive form. Ca2+ ions bind to Gla domains and help localise clots to site of damage

34
Q

What are Gla domains? What do they allow?

A

gamma-carboxyglutamate residues. Post translational modification of coagulation factors in liver. Addition of COOH groups to Glu residues to form carboxyglutamate (Gla). Allow interaction with sites of damage and bring together clotting factors.

35
Q

Which three things help ensure the clot is made at exactly the site of damage? Say how

A

Gla - target prothrombin to the site of damage
Kringle - help keep prothrombin in inactive form until needed
Ca2+ - binds to Gla domain helps localise clots to site of damage.

All ensure clotting factors are at site of damage.

36
Q

How does fibrinogen –> fibrin –> clot (2)

A

1) Thrombin cleaves fibrinopeptides A and B from central globular domain of fibrinogen
2) Globular domains at the C term ends of the beta and gamma chains then interact with exposed sequences at the N termini of the cleaved beta and alpha chains to form a fibrin mesh or clot

37
Q

How does this soft clot become a hard clot? Which two enzymes?

A

Further cross-linking - enzymes

Transglutaminase

38
Q

How is transglutaminase activated?

A

From a zymogen proteolytically cleaved by Thrombin

39
Q

Give an example of a disease caused by a problem with an allosteric activitor (regulates enzyme activity)

A

Haemophilia factor VII deficiency

40
Q

Which pathway extrinsic or intrinsic is self sustaining?

A

Intrinsic

41
Q

Is factor VIII a protease? How does it work?

A

No, activates other proteases - IX

42
Q

How is clotting amplified and accelerated?

A

Factor VIII increased by limiting proteolysis by thrombin and factor Xa - this positive feedback amplifies the clotting signal and accelerates clot formation.

43
Q

How do you treat Haemophilia?

A

Recombinant factor VIII

44
Q

How is the intrinsic pathway self-sustaining?

A

Allosteric activation by Thrombin + feedback on factor V, VIII and XI

45
Q

Defects in protein C can lead to what disease?

A

Thrombotic

46
Q

Which drug acts on AT3? What is the role of AT3?

A

Heparin

AT3 is a specific inhibitor- binds to thrombin (not when bound to thrombomodulin)

47
Q

How is a clot broken down? What is the basis for treatment of?

A

Plasmin - fibrinolysis to fibrin fragments

Of embolic stroke caused by blood clot

48
Q

Name 5 ways Thrombin is involved in the regulation of the coagulation cascade?

A
  • Catalyses fibrinogen - fibrin by cleaving A and B sites on fibrinogen (fibrinopeptides)
  • When thrombomodulin binds to thrombin it activates protein PKC which degrades V and VIII
  • Thrombin has a + allosteric effect on factors V VIII and XI
  • Thrombin activates protransglutaminase to transglutaminase which catalyses further cross linking to form ‘hard clot’
49
Q

What are the three stages that lead to stopping the clotting process?

A

1) Coagulation factor dilution in blood
2) AT3 binding to thrombin
3) Protein C degrades factors V and VIII - activated by Thrombomodulin

50
Q

What is the role of Thrombomodulin and Protein C?

A

Thrombomodulin binds to thrombin and activates Protein C which degrades factors V and VIII - stops clotting process