haemoglobin

Question 1

what are examples of allosteric modulators of haemoglobin

Answer 1

oxygen, H+, CO2, organic phosphates

they are also allosteric effectors of myoglobin

Question 2

where does oxygen bind to in Hb/Mb

Answer 2

oxygen binds reversibly to Fe(II), not Fe(III),

in haem group there is a four membered ring, each member has an N atom pointing towards the iron at the top there at 2 carboxyl groups which let it exist in relatively hydrophillic environment, bottom of the molecule is very hydrophobic

Question 3

how many subunits do Hb/Mb have

Answer 3

Hb: 4
Mb:1

Question 4

where are haem groups found

Answer 4

both Mb and Hb

Question 5

what is the P50, how does it compare for Mb and Hb

Answer 5

P50 is the pressure where oxygen saturation = 50%
Mb: 1 torr
Hb: 26 torr
(Mb has much higher affinity to oxygen than Hb)

Question 6

describe oxygen saturation curves for Hb and Mb

Answer 6

Hb: sigmoidal (does not bind O2 at low partial pressures)
Mb: hyperbolic

Question 7

how many molecules of oxygen can Hb bind to

Answer 7

4 since it has 4 harm groups, 1 in each subunit

Question 8

how do foetal and adult Hb compare

Answer 8

foetal has higher affinity than adult for oxygen

foetal contains 2 alpha subunits and 2 gamma subunits

adult contains 2 alpha subunits and 2 beta subunits

Question 9

how does to O2/CO2 blood equilibrium compare in the lungs to in other tissues

Answer 9

in lungs equilibrium is on side of CO2 removal and O2 uptake (higher conc of oxygen in air and lower CO2 conc in air)

in tissues equilibrium is on side of O2 release and solubilisation of CO2

Question 10

describe the reaction equation that carbonic anhydrase catalyses

describe the equation describeing reactions between protons, oxygen and Hb

combine these to equations to get an overall equation

how do these equilibria change in lungs and in tissues

Answer 10

= is a reversible reaction sign

HCO3- + H+ = H2CO3 = CO2 + H2O
(carbonic anhydrase catalyses reaction converting CO2 and H2O into H2CO3)

O2 + HHb+ = H+ + HbO2

overall reaction:
HHb+ + O2 + HCO3- = HbO2 + CO2 + H2O

in the lungs the equilibia for all the reactions favour right hand side, in the tissues they favour left hand side

Question 11

how does binding of oxygen to Hb affect its visual absorption spectra, how does this compare to Mb

Answer 11

oxygenated Hb is red, deoxyHb is purple

visual absorption spectra changes a lot upon binding of O2

Hb and Mb have very similar visual absorption spectra

Question 12

what is the physiological benefit of Hb’s O2 binding curve, what is the change in binding affinities called

Answer 12

benefit of sigmoidal binding affinity of Hb is that it has low affinity in respiring tissues so it can offload O2 efficiently, but high affinity in lungs so it can transport oxygen efficiently

change between weak binding and strong binding is known as allostericism

Question 13

how does binding of O2 effect affinity of Hb to O2

Answer 13

binding of first O2 molecule increases affinity of Hb to bind more O2, increases with more O2 binding

Question 14

what molecules decrease Hb’s affinity for O2

Answer 14

CO2, BPG, protons, chloride

when CO2 and BPG are added the affinity is reduced to that found in blood

affinity drops at lower pH due to actions of protons

chloride reduces Hb affinity but to lesser extent

Question 15

how does BPG affect Hbs affinity for O2, what is the process and how does this relate to foeatal Hb

Answer 15

decrease in affinity;

BPG has 5 negative charges, binds to Hb and causes oxygen to release

BPG consists of a glycerol 3 carbon chain with 2 phosphate groups on either end and one carboxyl group in the middle

in T state there is a hole in the middle of Hb, BPG binds to this central hole

BPG binds to his143, lys82 and his2 on each of the beta subunits (6total binding sites)

foetal Hb loses 2 positive charges by replacing its his143 with a ser143, decreasing its affinity for BPG and increasing the oxygen affinity

BPG is all negative charges which bind to positive charges

Question 16

what is the theory of cooperativity

Answer 16

allosteric enzymes show cooperative substrate binding, the degree of co-operativity is modulated by allosteric effectors

An allosteric inhibitor increases co-operativity to slow down the rate at a specific subtrate concentration

an allosteric activator decreases co-operativity, at high activator concentrations the Vo or rate of the enzyme against substrate concentration becomes hyperbolic

applies to Hb and cooperative enzymes

Question 17

what are the models concerning Hb binding to O2

Answer 17

concerted model, sequential model

Question 18

describe the concerted model

Answer 18

Hb can either exist in taut or relaxed state

taut (T) state is deoxyHb and relaxed (R) state is oxy-Hb

Hb is either all relaxed or not at all in concerted model, hybrid states do not exist, independent of how much oxygen is bound, the amount of oxygen bound simply shifts equilibria between T and R states

(applies to aspartate transcarboxylase as well)

Question 19

describe the sequential model

Answer 19

sigmoidal affinity behaviour of Hb is produced by substrate binding to a subunit, inducing the T to R transition in that subunit

the change in one subunit influences its neighbours

in this model hybrid states must occur, allosteric activators stabilise R state and inhibitors stabilise T state

there is equilibrium between T and R states

in sequential model the states from T to R are:

T4=RT3=R2T2=R3T=R4

each equilibria has a different K value

Question 20

what ligands promote R/T state in haemoglobin/myoglobin

Answer 20

haemoglobin T state: organic phosphates such as BPG, CO2, chloride and H+

R state: oxygen

Mb: nothing, Mb is not a cooperative protein and so does not have an R/T state

Question 21

what are symptoms of altitude sickness

Answer 21

dizziness, shortness of breath, headaches and nausea

Question 22

what is method for protein crystallography

Answer 22

a source of protein is taken from a natural source or expression system

it is purified before it is then crystallised, crystals are usually 0.1-1mm

x ray crystallography is then used

crystal is placed in capillary tube and sealed off at each end with buffer so crystal does not dry out

phases are then estminated using data which can be used to calculate an election density map, using this data you refine the preliminary atomic model to create a final atomic model

Question 23

what is the structure of myoglobin, how does it compare to Hb

Answer 23

one haem group surrounded by 8 alpha helices, the alpha helices create a hydrophobic pocket which the haem is contained in

function of alpha helices is to provide this pocket for hydrophobic haem

myoglobin and haemoglobin have 8 structurally similar alpha helices labelled A to H making 75% of the secondary structure. Helices A B and E are on top, F passes from top to bottom, G and H are on the bottom. The haem is sandwiched in the angle between helices E and F. E is longer than F.

A-H happens sequentially from the N terminus ending at the C terminus.

myoglobin contains 153 amino acids, 131 of which are in alpha helices

Question 24

what is the biochemical process that causes oxygen binding to increase Hbs affinity

Answer 24

haem binds to nitrogen atom of histidine residue called hisF8/ histadine F8

binding of oxygen causes slight conformational change in helix F which allows for the allosteric effect in Hb, this is transmitted through F8

when oxygen binds it pulls iron atom into the plane of the hae, the iron atom then pulls hisF8 and helix F moves with this

Question 25

how is oxygen access to haem controlled

Answer 25

a distal histidine, his E7 controls oxygen access, which is on E helix

Question 26

how does the subunit interface structure vary in T and R states

Answer 26

the alpha1beta2 interface switches from the T to R form on oxygenation. The “dove tailed” construction of this interface allows the subunits to readily adopt either of the two conformations.

in T state a salt bridge forms between aspartate G1 of beta subunit and tyrosine C7 of the alpha 1

in R state a salt bridge is formed between aspartate G1 of the alpha 1 and asparagine G4 of the beta 2

in R state the alpha1beta2 and alpha2beta1 dimers move relative to eachother by 15 degrees

Question 27

how do F,G and C helices relate

Answer 27

extensive subunit interactions occur between FG corners and C helices

the beta 2 FG corner interaction with the alpha C helix is the switch region

the flexible joint region is the interaction between the alpha 1 FG corner and beta 2 helix

the switch region is located below the beta2 C terminus and the joint region is located above the alpha1 C helix terminus

Question 28

what causes sickle cell anaemia

Answer 28

amino acid mutation from beta Glu6 (hydrophillic) to beta Val6 (hydrophobic)

Question 29

what is the sickling cycle and what is the molecular basis

Answer 29

(Hb-S is sickle cell Hb)

oxy-Hb-S becomes deoxy Hb-S

deoxy Hb-S precipitates as long fibres, causing sickling of erythrocytes

this causes blockage of the blood vessel, this creates a local region of low oxygen tension which leads to more deoxygenation and more sickling which can lead to a myocardial infarction

molecular basis: valine creates a hydrophobic stick patch on ends of Hb-S, deoxy Hb-S hydrophoic sites form, which the stick patches stick to creating a chain and causing aggregation

Question 30

what is thalassaemia

Answer 30

absence of a globin chain, either alpha or beta, usually occurs in mediterranean populations

alpha-thalasseaemia haemaglobin is made up of 4 identical beta chains where no or very little alpha chains are formed, the molecule shows non cooperativity and high oxygen affinity, behaves like 4 myoglobin molecules that are connected

Question 31

what is mechanism for increase in oxygen affinity after binding to Hb

Answer 31

there are 8 key salt bridges in haemoglobin, 2 between each subunit. This helps explain allostericism since breaking the first subunit off requires breaking 4 salt bridges, subunits 2 and 3 require breaking 2 salt bridges and the last one requires no breaking of salt bridges.
BPG creates more salt bridges between beta subunits thus making salt bridges harder to break.

Question 32

how are subunits in Hb structured

Answer 32

haemoglobin has 4 protein subunits, 2 alpha and 2 beta which associate as alpha1beta2 and alpha2beta1.

no contact between haem groups in Hb, signalling between haems for allosteric effect occurs through protein structure.

Question 33

what body changes occur at high altitude

Answer 33

the way Hb acclimitises to high altitude is to make more BPG which shifts saturation curve to the right which creates a difference in 37 of the oxygen it carries. Before this happens, at an alititude of 4500 metres it is only 30%, normally at sea level the value is 38%.