8.3.16 Lecture

Question 1

What are the four major differences between myoglobin and hemoglobin?

Answer 1

1. Location: Skeletal muscle (Mb) vs. Red blood cells (Hb) 2. Job: Oxygen storage for contraction (Mb) vs. Oxygen transport from lungs to tissues (Hb) 3. Structure: Monomer (Mb) vs. Tetramer (Hb) 4. Prosthetic group(s): 1 Heme-Fe2+ (Mb) vs. 4 Heme-Fe2+ (Hb)

Question 2

What is a prosthetic group?

Answer 2

A prosthetic group is a nonpolypeptide moiety that forms a functional part of a protein.

Question 3

Describe the secondary structure of myoglobin.

Answer 3

• All helical (8 helices, A-H)
• 153 amino acids
• Oxygen-binding heme group non-covalently bound to a pocket in the protein.

Question 4

Mb and Hb are ___% identical and ___% similar.

Answer 4

26; 59

Question 5

What are two differences between the secondary structures of Mb and Hb?

Answer 5

1. Hb N-terminus is longer and C-terminus is shorter 2. Hb has more hydrophobic residues on the surface to make contact with the three other subunits.

Question 6

Describe the secondary structure of hemoglobin.

Answer 6

Hemoglobin is composed of 2 alpha chains and 2 beta chains. Alpha1Beta1 and Alpha2Beta2 each act as a dimer. These two dimers form the tetramer.

Question 7

What are the two types of hemoglobinopathies (hereditary disorders)?

Answer 7

1. Structural variants (altered amino acid sequence) 2. Thalassemias (decreased abundance of one or more globin chains)

Question 8

Describe the pathology of sickle cell disease (HbS).

Answer 8

Oxygenated blood appears normal; deoxygenated blood assumes the sickle cell shape. These block capillaries, preventing blood flow to the tissues, which results in local ischemia and a painful sickling crisis. The RBCs also have weak membranes, which can break (hemolysis), leading to a decrease in RBC and to anemia.

Question 9

What is the mutation in HbS? How does this change the structure?

Answer 9

Glu6Val; this amino acid is found on the surface of beta chains and the mutation changes it from a hydrophilic amino acid to a hydrophobic one. Val binds to a normal hydrophobic patch in Hb, leading to aggregation, polymerization, and ultimately deformation.

Question 10

What type of genetic disease is HbS?

Answer 10

Autosomal recessive

Question 11

If you are heterozygous for HbS, you have the sickle cell ___. If you are homozygous for HbS, you have the sickle cell ___.

Answer 11

Trait; disease

Question 12

There is a geographically higher frequency of sickle cell trait in…

Answer 12

…areas where malaria is common; those with the trait are resistant to malaria.

Question 13

Describe the structure of heme.

Answer 13

• Planar porphyrin structure
• Ferrous (Fe2+) ion in the middle
• N from pyrrole groups make coordinate covalent bonds with Fe2+
• One edge of heme has hydrophobic groups; the other has hydrophilic proprionic acid groups
• Fe2+ binds oxygen

Question 14

What happens if the ferrous ion is oxidized?

Answer 14

It is changed to Fe3+ (ferric ion), which cannot bind oxygen. This is known as methemoglobin.

Question 15

Where is the heme binding site found?

Answer 15

Between the E and F helices

Question 16

What mutation occurs in Hb Hammersmith? How does this affect Hb?

Answer 16

Phe42Ser; Ser is smaller and hydrophilic, which causes the heme to slip out of the pocket. Hb becomes unstable, aggregates, and precipitates. It does not polymerize.

Question 17

Describe the process of oxygen binding to heme.

Answer 17

1. F8His (proximal His) makes a 5th covalent bond with Fe2+. 2. O2 makes 6th bond to Fe2+ 3. E7His (distal His) hydrogen bonds with the oxygen

Question 18

What are the two conformations of heme?

Answer 18

1. Deoxygenated (found in the tissues): T-conformation 2. Oxygenated (found in the lungs): R-conformation

Question 19

Which conformation of heme has a greater oxygen affinity?

Answer 19

R (150-300x greater than T)

Question 20

As the concentration of oxygen increases, oxygen begins to bind to hemoglobin. Conformational changes occur in one subunit and these are transmitted to the others. The oxygen affinity increases. What is this called?

Answer 20

Positive cooperativity

Question 21

Describe the mechanism of positive cooperativity in hemoglobin.

Answer 21

1. In the T-conformation, Fe2+ is slightly out of the plane of heme. 2. Oxygen binds to Fe2+, bringing it more into the plane. The proximal histidine also moves, as it is attached to oxygen. 3. The F helix changes conformation, which changes the FG corner. 4. The FG corner interacts with the C helix of a neighboring subunit through non-covalent interactions. 5. The C helix moves toward the R-conformation.

Question 22

Interactions between hemoglobin subunits occur primarily between ___ and ___.

Answer 22

Alpha1Beta2 and Alpha2Beta1

Question 23

What is Hb Kempsey?

Answer 23

Hemoglobinopathy caused by Asp99Asn, which is found at the FG corner/Alpha1Beta2 interface. This mutation locks hemoglobin in a high oxygen-affinity structure (R). It cannot release oxygen.

Question 24

What is oxygen saturation?

Answer 24

The percent or fraction of Mb or Hb that has oxygen bound.

Question 25

What is pO2?

Answer 25

The concentration of oxygen given as a partial pressure (torr).

Question 26

What is p50?

Answer 26

The pO2 where 50% of Mb or Hb has oxygen bound.

Question 27

Between Mb and Hb, which has a higher oxygen affinity?

Answer 27

Mb

Question 28

Draw the Oxygen Binding Curves for Mb and Hb.

Answer 28

Question 29

Cooperativity can be measured by the ___ of oxygen needed to go from 10% to 90% saturation.

Answer 29

Fold-change

Question 30

Smaller fold-change indicates ___ cooperativity.

Answer 30

Higher

Question 31

What is the Hill coefficient?

Answer 31

1 = no cooperativity \<1 = negative cooperativity \>1 = positive cooperativity

Question 32

How does pH affect hemoglobin/oxygen binding?

Answer 32

When T binds oxygen to become R, ~1-2 H+ are released. As the number of hydrogen ions increases, the pH decreases (becomes more acidic), shifting the reaction to the left. This increases oxygen release. The oxygen-binding curve shifts to the right. Hb + 4O2 \<\> Hb(O2)4 + nH+

Question 33

In the tissues, metabolizing cells produce ___ and ___ acids, decreasing the pH. What does this do?

Answer 33

Lactic; carbonic; enhances O2 release by decreasing the pH, increasing p50.

Question 34

Where is BPG made? Describe its structure.

Answer 34

Made in RBC, has 2 phosphates and 5 negative charges.

Question 35

BPG increases under ___ conditions.

Answer 35

Hypoxic (anemia, high altitude, etc.)

Question 36

How does BPG bind to Hb?

Answer 36

BPG fits into the positively charged Beta1Beta2 interface of the T conformation (due to its 5 negative charges), stabilizing the conformation. It does not bind to oxygenated Hb.

Question 37

How does BPG affect hemoglobin/oxygen?

Answer 37

As BPG increases, the reactions shifts to the left, increasing the amount of Hb in the T conformation. This increases p50 and increases oxygen release to the tissues.

Question 38

What are some of the different types of globin genes and their chains?

Answer 38

1. HbA (alpha and beta chains): adult Hb 2. HbGower1/HbGower2/HbPortland (zeta, eta, and gamma chains): Embryonic Hb 3. HbF (alpha and gamma chains); fetal Hb

Question 39

How is HbF different than HbA?

Answer 39

HbF has a high oxygen affinity and pulls oxygen from the mother's deoxygenated HbA.

Question 40

\_\_\_ chains are made early in development and continue to develop through adulthood. ___ chains are made at a low level prior to birth, and increase after birth. ___ chains are high in the fetus and decrease post-birth.

Answer 40

Alpha; beta; gamma

Question 41

What is a thalassemia?

Answer 41

An imbalance of alpha and beta globin subunits; carrier resistant to malaria

Question 42

What are the two major types of thalassemia?

Answer 42

Alpha-thalassemia: mostly alpha-globin gene deletion Beta-thalassemia: mostly single base pair mutations, leading to decreased or defective mRNA.