Hemoglobin, an Allosteric Protein

Question 1

What is Hemoglobin? What kind of protein is it?

Answer 1

Hb- a red blood cell protein that carries oxygen from lungs to tissues and also helps transport CO2 and H ions back to lungs.
important allosteric protein that has cooperativity in O2 binding and release.

Question 2

Describe the structure of Hb and its importance.

Answer 2

Tetramer (quatenary structure) made of 2 alpha-beta dimers.

Question 3

Distinguish between myoglobin and hemoglobin. describe the binding properties for each.

Answer 3

myoglobin- protein seen in muscle cells. binds oxygen, but NOT cooperative; HIGHER affinity for O2 (holds on tight)
Hemoglobin- protein in RBC’s that is COOPERATIVE in O2 binding and release.
LOWER affinity for O2 (releases oxygen more readily)

Question 4

Explain the concept of cooperativity and its importance in Hb.

Answer 4

Cooperativity- the ability of all of Hb’s 4 subunits to change conformation due to Oxygen to binding one subunit out of Hb’s 4 subunits which increases affinity for O2 bind to other 3 subunits .

Question 5

Describe the structure of myoglobin. What structural feature does myoglobin have in common with Hemoglobin?

Answer 5

Single polypeptide chain made of mainly alpha helixes, turns to form globular structure. Has no subunits.
Hb and Mb both bind oxygen at heme group (prosthetic group, containing Fe) and form globin structures

Question 6

Describe the structure of heme group. where does iron atom form in the structure?

Answer 6

Heme group- Fe iron, polyphorin ring, 4 N atoms, 4 Methyl groups,

Question 7

distinguish between 5th and 6th coordination sites in heme group? Why are they important?

Answer 7

5th coordination site- ring of histidine is in pocket, Fe iron moves up into polyphoryn ring.
6th coordination site- nothing there, Fe iron can move

Question 8

What enzyme facilitates formation of bicarbonate ions.

Answer 8

Carbonic anhydrase

Question 9

Describe what occurs in T and R states depending on oxygen binding. How does it affect iron atom?

Answer 9

T and R states are conformations of Hb. T- tense state (not active, high affinity for O2), R state (very active, low affinity for O2).

Question 10

How does 2, 3 BPG affect T and R state and Hb

Answer 10

2, 3 BPG enhances T state, lowering affinity for Hb to bind to O2.

Question 11

what makes fetal hemoglobin different from Mother’s hemoglobin? how does it affect affinity for Oxygen?

Answer 11

Fetal hemoglobin is not affected by 2,3 BPG that will affect mother’s Hb and lower affinity to bind O2 to Hb. therefore Fetal hemoglobin will be able to have increasing affinity of binding o2 to Hb.

Question 12

What is the Bohr Effect?

Answer 12

Bohr Effect- how interaction of pH will alter binding affinity for O2 in Hb.

Question 13

What is sickle cell anemia? Explain the mutation in Hb.

Answer 13

Sickle cell anemia- genetic disease caused by mutation resulting in substitution of Valine for glutamate at position 6 of Beta chains
Mutation in Hb: In HbS, acidic glutamate residue replaced by nonpolar Valine residue.
Results in solubility reduced for deoxygenated form of Hb. The Valine will interact with hydrophobic pocket (site of Phenylalanine b85 and Leucine B88)on another deoxygenated beta chain which creates aggregation of deoxy Hb into insoluble fibers.

Question 14

Differentiate between sickle cell anemia and sickle cell trait.

Answer 14

Sickle cell anemia- disease due to both parents having abnormal sickle cell allele and passing down 2 abnormal alleles to offspring.
Sickle cell trait- have one sickle cell allele (one mutated allele), one normal allele People are not really affected by this allele, no life-threatening disease. Individuals are asymptomatic, just CASE STUDY.

Question 15

why is there a high incidence of HbS allele in Africans?

Answer 15

Hbs allelle in RBC are resistant to malaria which is very prevalent in West African region. Heterozygous trait being carrier protects people living in these regions from getting malaria.

Question 16

What is Thalassemia? Differentiate between alpha thalassemia and beta thalassemia.

Answer 16

Thalassemia- loss or substantial reduction of Single hemoglobin chain
Alpha thalassemia- alpha chain not produced. Tetramers of the B chain form (HBH) and bind Oxygen with high affinity, but NO COOPERATIVITY (hence low O2 release to tissues)
Beta thalassemia- beta chain not produced. The alpha chains AGGREGATE and precipitate, leading to loss of RBC and anemia.

Question 17

Discuss how O2 is transported from lungs to tissues based on partial pressure.

Answer 17

O2 transported to lungs with high partial pressure (100 torr) to the tissues (low partial pressure of 20 torr)

Question 18

Describe the change in Oxygen saturation from lungs to tissues in Hb vs. Mb (Myoglobin)

Answer 18

In Hb, the lungs nearly reach saturation with 98 % of O2 binding site occupied. when Hb in tissues, O2 saturation drops to 32% which results in 66% of O2 binding sites releasing O2 in tissues.
Myoglobin on other hand only 7% of binding sites release O2

Question 19

*Describe the effect of carbon monoxide (CO) on hemoglobin. Why is CO toxic?

Answer 19

CO is very deadly gas (colorless, odorless). CO binds very tightly to Hb at oxygen binding site, even binding 200 times more tightly as Oxygen to Hb.
Even if only one of the four O2 binding sites on Hb is occupied by CO and other three are bound to O2, Oxygen will still not be released (hence toxicity of CO) .

Question 20

*How do hydrogen ions (i.e. pH) and carbon dioxide affect the allosteric nature of hemoglobin?

Answer 20

H+ Ions and CO2 that are produced by actively respiring tissues enhance oxygen release by Hb.
O2 affinity of Hb decreases as pH decreases from 7.4 in lungs (100 torr) to pH of 7,2 in tissues (20 torr). This leads to release of O2, to 77% carrying capacity (more than 2,3 BPG)
pH affect on oxygen release is due to ionic bond interaction in deoxyhb, where 3 aa residues form salt bridges that stabilize T quaternary structure of Hb.
One of salt bridges depend on added proton on His B146 which is favored by proximity with negative charge on aspartate B94. Other salt bridge or ionic bond forms between carboxyl terminus of His B146 and Lysine Alpha 40 which stabilizes T state.
The decrease in pH, hence the two salt bridges allow for the deoxhb to be stabilized and leading to release in more o2.
Also, with CO2, Hb reacts by decreasing affinty for oxygen and releasing more o2 in tissues with high Co2 [ }. In presence of Co2, 40 torr, and pH of 7.2 Oxygen released is 90% carrying capacity (higher than all of the rest of regulators). Co2 stabilizes Deoxyhb by reacting with terminal amino groups to form carbamate groups which are negatively charged. The carbamate forms salt bridges that stabilize T state
Hence CO2 and H+ are heterotropic regulators of O2 binding by hemoglobin
low pH allows formation of ionic interations that stabilze T state and enhance O2 release.

Question 21

*Describe the difference between adult hemoglobin and fetal hemoglobin? Why is there a
difference?

Answer 21

fetal hemoglobin- has lower affinity for binding 2,3 BPG, hence has a higher O2 affinity that it needs to have when mother is releasing O2. That way, fetus will be able to receive enough oxygen from mother to survive.
Adult hemoglobin- can bind to 2,3 BPG better than fetus hb, so it’s O2 affinity will be reduced by 2,3 BPG so it can release oxygen.
Difference is affinity for both 2,3 BPG and oxygen (fetus has lower 2,3 BPG affinity, and higher O2 affinity)
Also Fetus hb have 2 alpha subunits and 2 gamma subunits; substitution in gene where serine replaces histidine 143, removing 2 positive charges away from 2,3 BPG and lowering its affinity for 2,3 BPG.
Adult hb have 2 alpha, 2 beta subunits

Question 22

*How does 2,3-bisphosphoglycerate (2,3-BPG) function?

Answer 22

2,3 BPG is an allosteric regulator for Hb. When 2,3 BPG binds to Hb in RBC it will reduce the oxygen affinity, so 66% of O2 will be released into the tissues.
However, unlike Hb, pure HB (no 2,3 BPG) will not release as much O2 to tissues (only 8%), hence why this regulator is important.
2, 3 BPG stabilizes T state of hemoglobin and thus facilitates release of O2 ( It will preferentially bind to deoxyhb, reducing its affinity, releasing o2)

Question 23

*explain the cooperative nature of hemoglobin in binding oxygen.

Answer 23

When oxygen binds to one of hemoglobin’s four binding sites, it increases the affinity of Oxygen to three of the other binding sites (O2 binding increases affinity of hb for more O2) .

Question 24

*What is a heme group?

Answer 24

prosthetic group that allows Hb and Mb to bind O2.
it has protoporphyrin structure, central Fe ion in ferrous (Fe^2+) connected by 4 nitrogens, 4 pyrroles, 4 methyls, 2 vinyl and 2 propionate groups.
I

Question 25

*Which one of the globins is more efficient in binding oxygen? Why?

Answer 25

Hemoglobin because it is an allosteric protein that has high cooperative binding and release.

Question 26

*Understand the distinction between myoglobin and hemoglobin. What is the difference between
a hyperbolic curve and a sigmoidal curve?

Answer 26

Myoglobin-single polypeptide chain composed mainly of alpha helices, binds O2 at heme (but not cooperatively), does not release as much O2.hyperbolic shape; seen in muscle cells; ONE O2 binding site
Hemoglobin- multi-subunit allosteric protein that displays cooperativity in oxygen binding and release, carries O2 from lungs (100 torr) to tissues (200 torr); seen in red blood cells. sigmoidal shape; 4 O2 binding sites.
Sigmoidal- Hb releases O2 more readily.
hyperbolic- myoglobin has higher affinity to O2. will pick up o2 more readily and will tend to hold on to O2.

Question 27

How many additional bonds can Iron from heme group form? explain this process.

Answer 27

Fe can form two additional bonds, called 5th and 6th coordination sites.
5th coordination sites- occupied by imidazole ring of histidine-called proximal histidine
6th coordination site- binds oxygen
When oxygen binds to site, iron will move into plane of protoporphyrin ring (proximal histidine which is component of alpha helix will move with the iron.
creates conformational change and is communicated with other subunits so that 2 aB dimers rotate with respect to one another- form R state .

Question 28

What are two forms of Myoglobin?

Answer 28

Can have deoxymyoglobin- O2 free
oxyhemoglobin- o2 bound
also for Hb as well deoxyhb and oxyHb

Question 29

What physical property does heme group provide?

Answer 29

It gives red color of muscle cells and blood seen in Mb an Hb.

Question 30

Compare and contrast what occurs in the sixth coordination site for deoxyhemoglobin vs oxymyoglobin

Answer 30

In sixth site in deoxyhemoglobin, the site is unoccupied (position available for O2 binding) The Iron group(in ferrous form Fe^2+) is outside of the plane in porophoryn ring because it is too large to fit the well-defined hole in the ring.
In Oxyhemoglobin, Oxygen will be bound to sixth coordination site and will allow rearrange electrons in heme iron ion so it will be smaller and now fit into porporhyn plane (O2 stablized with H-bond with distal histidine)

Question 31

What is the purpose of the distal histidine?

Answer 31

The distal histidine is located on opposite side of proximal histidine.
distal histidine prevents oxidation of heme iron (Fe^2+) into ferric ion, (Fe^3+) since it cannot bind Oxygen and will reduce ability of CO to bind to heme.

Question 32

Elaborate more on the quaternary structure of Hb? how many dimers are present? Heme groups?

Answer 32

Hb is a tetramer consisting of 2 alpha subunits and 2 Beta subunits. each subunit has a bound heme (total 4 heme groups for Hb).
Quaternary structure described as pair of IDENTICAL ALPHA-BETA Dimers (alB1, a2B2)

Question 33

How are Hb dimers linked in deoxyhemoglobin vs oxyhemoglobin?

Answer 33

In deoxyhemoglobin, the Alpha Beta dimers are linked by extensive interface (leaving central cavity (hole) in center of globin structure. Also, deoxyhb, corresponds to T state of allosteric enzymes.
In Oxyhemoglobin,- where oxygen binding occurs, transition from T state (from deoxyhb) to R state occurs
iron moves ito plane of heme, and proximal histidine moves with iron. This structural change is communicated to other subunits so that the Alpha Beta dimers rotate with one another form R state. Central cavity will close.

Question 34

How does 2,3 BPG binding accomplished?

Answer 34

2,3 BPG binds to a pocket in the HB tetramer that exits only when hb is in T state.
Binding occurs: ionic binding interaction between negative charges on 2,3 BPG and positively charged groups on each Beta chain (lys 82, His 143, His 2)

Question 35

How does Hb play a role with Co2, H+ and O2

Answer 35

Hb can transport Oxygen but also H+ and Co2 as it carries these last 2 ions into blood in lungs.

Question 36

What accounts for the major transport of Co2 and H+ in the blood?

Answer 36

Co2 and H+ is mainly transported through blood in lungs as bicarbonate. Carbonic anhydrase, an enzyme will faciitate bicarbonate ions that is produced in RBC and released in blood plasma.

Question 37

What are three factors that stabilze deoxy form of Hb?

Answer 37

1. 2,3 BPG binding
2. Ionic bonds between acidic and basic amino acids
3. Ionic bonds between amino-terminal carbamate

Question 38

Differentiate between sickle cell anemia and Thalassemia

Answer 38

Sickle cell anemia- genetic disorder due to mutation of single specific amino acid (substitute Valine for glutamate)
Thalassemia- blood disorder due to substitution or huge reduction of hemoglobin chain.

Question 39

Describe the incidence of sickle cell anemia in African Americans and why there is a much higher incidence in West Africa.

Answer 39

incidence of HbS allele is 4 in 1000 African Americans
incidence of HbS is 1 in 100 West Africans.
High incidence specifically in West Africa due to high prevalence of malaria in the continent and many people who have sickle cell trait (one mutated allele) , are resistant to malaria (and will likely survive and reproduce)
Heterozygous trait for HbS more resistant to malaria.