Hemoglobin Flashcards
1
Q
life needs oxygen
A
- most living organisms need oxygen to carry out basic metabolic functions-used for electron transport chain
- rate of o2 transport in tissues is inversely proportional to the square of the distance it must diffuse-makes diffusion rate too slow to support life thru tissue thicker than 1 mm
- evolution of larger organisms required to acquisition of oxygen carriers and circulatory systems-b/c o2 not soluble in water
- Hg is predominant-10 mM Hb monomer binds 10 mM o2 (compared to less than 1/4 mM in air sat water)
- Mg is intracellular o2 transport and storage protein
2
Q
oxygen transport-the big picture
A
- Hg just one step
- need o2 for oxidative phosphorylation
- ultimate destination is mito-cytochrome oxidase, Km <1 torr, which means it will run at full speed for any oxygen concentration above a few torr (2.5-5 in myocytes)
- o2 from atm (760 torr) is energetically downhill, need to speed it up
- Hb carries o2 from alveoli (100 torr) to tissues (20 torr)
- steepest gradient b/n red cell in cap and surface of myocyte-20 to 5 torr in 2 um or less
3
Q
myoglobin folding
A
- helical protein with 8 helices and a heme
- heme binds 02
- 153 aa
- similar to subunits of Hb, only 18% identical in primary sequence
- globular shape, 44x44x25 angstroms
- intracellular transport and temporary storage of oxygen needed for aerobic metabolism
- highly abundant in muscle cells
4
Q
Quaternary Structure of hemoglobin
A
- tetrameric protein with a2b2 (dimer of ab protomers, ab dimer is protomer)
- globular shape 64x55x50 angstroms
- each a and b subunit is bound non-covalently to a prosthetic heme group
- Hb molecule has 4 hemes that hold 4 o2 molecules (8 o atoms)
- B units toward front in diagram
5
Q
ApoHb
A
empty Hb tetramer consisting of a1b1 a2b2 subunits
6
Q
holoHb
A
- Hb
- ApoHb + 4 hemes
7
Q
assembly of hemoglobin
A
- assembles first into ab heterodimers=protomers
- two of the protomers come together to form the tetramer
- association is loose and flexible
- rapid eq b/n dimers and tetramer (favors tetramer)
- two different ways of associating: T and R
- T has lower affinity for o2, R has higher
- basis for coopertivity resulting in sigmoid binding curve
- protomers rotate 15 degrees b/n T and R
8
Q
heme
A
- porphyrin ring
- Fe-protoporphyrin IX, porphyrin with side chains
- heterocyclic ring systan, tetrapyrrole-4 pyrrole rings linked by bridging carbons
- nitrogens of each pyrrole face inwards to the center, creating a metal chelating site where Fe binds
- 4 methyl, 2 vinyl, 2 propionate substituent groups
- Fe likes hexacoordinate, in heme 4 ligands are provided by 4 N of the porphyrin ring (equitorial). the 5th and 6th ligand would be in front of and behind heme plane (axial).
- heme proteins will bind by providing aa which act as the axial ligands
9
Q
deoxy Hb
A
- Hb with no oxygen
- the iron is 5 coordinate with 5 N, 4 from pyrroles and other from proximal His
- electronic state Fe (II)-ferrous
- distal His in space behind
10
Q
o2 binding
A
- o2 binds the heme iron closer to the distal His
- Fe is hexa-coordinate-5 N and 1 O2
- o2 binds to distal His too
11
Q
o2 binding 2
A
- dramatic consequences for the properties of Hb
- deoxyHb is dark red while oxyHb is bright red
- small molecules like CO, NO, h2S bind heme with higher affinity-poison
- ferrous to ferric- makes methemoglobin or metmyoglobin-brown red- doesnt bind o2
- see slide-o2 can bind without affecting oxidation state
12
Q
clarification
A
- oxidation is redox state-ferrous or ferric
- oxygenation is whether or not oxygen is present
- coordination state refers to number of atoms bound to Fe- 5 or 6 depending on o2 binding
- oxidation of Fe in oxyHb is complicated, may be some resonance to ferric and a superoxide
13
Q
oxygen dissociation curves
A
- o2 is 20 torr in venous blood
- 100 torr in arterial blood
- 158 in atm
14
Q
P50
A
pressure at which binding is half maximal
-pressure value at which 50% of maximal o2 load released
15
Q
Mb
A
- hyperbolic curve
- P50 2.6- much higher affinity than Hb- makes sense, want Hb to give it up then
16
Q
Hb
A
- sigmoidal curve, coopertivity b/n o2 binding sites
- P5- 26- lower affinity than Mb at lower concentrations- want that
17
Q
coopertivity
A
- convolution of 2 molecular states
- high affinity of R state is high hyperbolic
- low affinity of T state is low hyperbolic
- curve mimics T and R and connects the middle
- o2 drives T-R conversion- one o2 binds means more likely to go to R and more will bind
- at high concentration, curve is closer to R-saturated; high affinity
- at low concentration- curve is closer to T- low affinity
18
Q
hemoglobin in vivo
A
- allosteric interaction
- binding of o2 to one site increases affinity for other binding sites on same protein
- effectors can decrease affinity for o2
- results from quaternary structure, which is in eq b/n T and R
- stripped purified Hb has a higher affinity for o2 than whole Hb
- effectors can be pos or neg
- positive effector is o2-stabilize R state
- neg is BPG (decreases affinity), CO2, H+, Cl- (inc affinity in lungs (R) and decrease in tissues)-stabilize T state
- T in muscles-releases o2
- R in lungs, gains o2
19
Q
allostery
A
- effect of something happening at another site on the active site
- other site can be another active site in a multimeric protein or it can be a completely different type of site, binding the same or another molecule as in allosteric regulation of PFK by ATP
20
Q
coopertivity
A
- type of allostery in which what is happening at one site promotes the same thing happening at another identical site like Hb
- requires having multiple sets of the same kind, generally a property of multimeric proteins
21
Q
positive effectors
A
- stabilize R state
- shift dissociation curve to left-increase affinity
22
Q
negative effectors
A
- stabilize T state
- shift curve to right-decrease affinity
23
Q
BPG
A
-present in whole blood
-decreased affinity for o2 by binding to heme and stabilizing T state (deoxyHB) (binds weakly to oxyHb)
-shifts curve to right
-leads to more release of o2 in respiring tissues
P50 is 3 torr without BPG- wouldn’t ever let go of o2, even in venous blood
-arterial blood po2 is 100 torr- Hb is 95% saturated
-venous blood po2 is 43 torr-Gb is 43% saturated
-Hb with BPG is an efficient o2 carrier and unloads 52% of o2 in capillaries
24
Q
BPG binding
A
- binds at tetramer interface and interacts with several lysines, histidines, and N termini
- stabilizes T state
25
Q
changing BPG [ ]
A
- acclimation to high altitude have altered BPG levels
- shifts curve to right even more (more BPG)-more o2 unloading because P50 is 31(takes longer to get to half sat)- lower affinity means more release of o2
- change in tissues is greater than in lungs
26
Q
Bohr Effect
A
- oxygenation of Hb makes it a stronger acid
- lungs have lower CO2, higher pH-lower H-Hb has higher affinity for o2, stabilizes R state
- tissues have higher CO2, lower pH-higher H-lower affinity for o2. stabilizes T state
- look at slide for reaction-low H in solution drives reaction towards more H in environment and o2 onto Hb (tissues go other way)
- in RBCs, carbonic anhydrase speeds reaction up
27
Q
CO2 regulates other ways
A
- combines reversibly with N terminal aa of blood proteins to form carbamates
- leads to decreasing affinity in tissues, more o2 out
- CO2 stabilizes T state (binds more readily with deoxy Hb)
28
Q
fetal hemoglobin
A
- a2gamma
- gamma has higher affinity than beta
- P50 is 15
- deoxyHbF has lower affinity for BPG, more R state at lower o2 [ ]
29
Q
HbA2
A
-3% of Hb in RBCs has 2 alpha chains and 2 delta chains
30
Q
variants
A
- there are almost 900 naturally occurring variants of Hb known, >90% from a single aa substitution in one of the globin polypeptide chains
- ~300,000 ppl with serious disorders born every year
- ~5% of worlds pop have an inherited variant Hb
31
Q
changes in surface residues
A
- mutations are usually innocuous except sickle cell
- Glu to Val=HbS
- leads to aggregation of B dimers due to hydrophobic binding, only one of the 2 Val6B contacts neighbor
- hemolytic anemia
- heteros protected from malaria, RBCs shorter lifetime
32
Q
changes in internally located residues
A
- destabilize Hb structure and or alter its o2 binding dunction
- hemolytic anemia
33
Q
changes in stabilizing methemoglobin
A
- usually near o2 binding site, favor heme oxidation
- can’t bind oxygen
- methemoglobinemia
- promotes conformational change and stabilizes R state, don’t release oxygen in tissue
- bluish skin
34
Q
linus pauling
A
-coined the term molecular disease
35
Q
HbC
A
- subunit a2b2
- glu to lys
- doesn’t polymerize
- protects against malaria
36
Q
HbH
A
- b4
- binds o2 very tightly and non coopertively
- inefficient o2 delivery in tissues
37
Q
HbBarts
A
- gamma4
- high affinity non coopertive binding to o2, poor o2 delivery
38
Q
detect forms of Hb
A
-native gel electrophoresis
