Chapter 7: Hemoglobin and Myoglobin pt.2

Question 1

protein family

Answer 1

group of evolutionary related proteins

-share a common ancestor and have similar 3D structures, functions, have significant sequence similarity

Question 2

Myoglobin (Mb)

Answer 2

-monomer
-have 8 alpha-helices (A-H)-both
*onlynhave 24/141 AA identical to Hb
*Mb is very similar to 3D structure of beta subunit of hemoglobin

Question 3

Hemoglobin (Hb)

Answer 3

-tetramer of 4 Mb like subunits
-have 8 alpha-helices (A-H)-both

Question 4

Yo2 curves for Myoglobin (Mb)

Answer 4

-hyperbola curve: no cooperativity

Question 5

Yo2 curves for hemoglobin (Hb)

Answer 5

-sigmoidal curve: cooperativity

Question 6

cooperativity/ allosteric effect

Answer 6

-allostery= the other site
-binding at one site affects binding at others
-allosterism req with protein with >1 subunit
-S curve indicates cooperativity (=allosterism)

Question 7

Homotropic allosteric interaction

Answer 7

effector and ligand regulated by the effector are the same molecule
*part of equation

Question 8

Heterotropic allosteric interaction

Answer 8

effector and ligand are different molecule
*effector is not in rxn

Question 9

Activator

Answer 9

shifts equilibrium towards R-state
=positive allosteric interaction

Question 10

inhibitor

Answer 10

shifts equilibrium towards T-state
=negative allosteric interaction

Question 11

Heterotropic inhibitors for hemoglobin

Answer 11

-BPG
-H+
-Co2
-Cl-

Question 12

homotropic effector for hemoglobin

Answer 12

O2

Question 13

The Bohr effect Summary

Answer 13

-increasing [CO2], increasing H+, decreasing pH, increasing temp. = releases O2 from Hb
*curve shifts to the right
*low affinity for O2

Question 14

what does the curve, temp, CO2, and pH look like when there is a high affinity for O2

Answer 14

-pH increases
-CO2 decreases
-temperature decreases

Question 15

Where on T-state do the Bohr protons bind?

Answer 15

on the N-terminal and His 146-beta (amide)

• H-bonds and ion pairs in the T-state are not present in the R-State
• T to R transition changes the pka’s of these groups causing them to release their coordinated H+
  *N-term needs to be protonated to be an ion pair
  *surroundings can effect pka of WA/WB of groups

Hb (t-state)+ O2 = Hb(r-state)O2 + H+
*H+ (bohr protons) released when Hb binds O2

Question 16

N-terminal amino groups are responsible for how much of the bohr effect?

Answer 16

20%-30%

Question 17

His 146 beta accounts for how much of the Bohr effect (has a NH group)

Answer 17

40%

Question 18

Bohr effect CO2: the carbonate rxn

Answer 18

-CO2 can react to form carbamates with the N-terminal amino groups of blood proteins
*CO2 reacts with the R-state Hb to create a T-State HB with carbonate
*Carbonate only in T-state

Question 19

Bohr effect CO2: CO2 transport

Answer 19

H2O+CO2<–>H2CO3<–>HCO3- + H+

-when there is more CO2 then H+ released increases so pH in blood decreases
*means that Hb is dumping off oxygen
*favors deoxy-Hb (t-state) = CAPILLARIES

-when there is less CO2 then H+ in blood decreases which means the pH is blood increases
* means that Hb is binding oxygen
*favors Oxy-Hb (r-state) = LUNGS

Question 20

D-2,3-bisphosphoglycerate (2,3-BPG) basic summary

Answer 20

-also known as BPG, 2,3-diphosphoglycerate, 2,3-DPG

• MOST ABUNDANT organic phosphate in blood
  -occurs in red blood cells as a HETEROTROPHIC INHIBITOR

-inhibits oxygen from binding
-curve goes towards x-axis

Question 21

2,3-BPG: where does it bind and what it does?

Answer 21

-binds to the central cavity of T-state and stabilizes it and “fine tunes” Hb’s O2 binding capability
*can’t bind the R-state
*causes HB to release O2

-gives Hb a P50 of 26 torr
*without it it only has 12 torr

Question 22

2,3-BPG and altitude

Answer 22

-body increases BPG production in higher altitudes
-restores 37% release of O2

Question 23

Allosteric effector: Cl-

Answer 23

-heterotrophic effector
- Cl- floods into RBCs to neutralize the electrostatic potential
-Cl- binds to deoxy Hb
*causes O2 to be released (t State)

Question 24

why are Hb’s and Mb’s called honorary enzymes

Answer 24

they bind O2 like how a enzyme binds a substrate

Question 25

Hill equation

Answer 25

YO2= pO2/P50+pO2 Ys=[S]^n/Kd+[S]^n *S=ligand/substrate *E=enzyme/protein *n=number of S molecules bound

Question 26

HIll plot

Answer 26

-slope of the line shows the degree of cooperativity -nH is experimentally determined -nH< n actual usually -nH= n actual when all ligand binds simultaneously

Question 27

1.n=1 2.n>1 3.n<1

Answer 27

1. non-cooperative 2. positive coop 3. negative coop

Question 28

hemoglobin cooperativity

Answer 28

about 3 -when the first O2 binds, the rest bind and when 1 is released the rest are released

Question 29

Monod, Wyman, Changeux

Answer 29

-MWC MODEL: model for allosteric proteins - equilibrium btwn T and R states -ligand can bind to T or R state conformation -all subunits have to be in the SAME STATE -in ABSENCE of ligand, equilibrium favors T state -Ligand binding shifts equilibrium toward R state -Limitation: only models positive cooperativity

Question 30

Koshland-Nemethy-Filmer

Answer 30

-found the Sequential (induced fit) model for allosteric protein -KNF model -subunits can exists in R or T conformations but can also change conformation independently on binding of a ligand -PRESENCE of R-site induces increased affinity in adjacent sites -complete T --> R transition is a sequential process *accounts for both positive and negative cooperativity

Question 31

Perutz mech for HB allostery

Answer 31

look at slide 16 for mech -basically once one oxygen binds it shifts from t-state to r-state and then all the other subunits get bonded to oxygen

Question 32

Mutation on hemoglobin (methemoglobin and cyanosis)

Answer 32

- when mutations occur in germ line they are passed to offspring *inheritable or genetic diseases -sickle cell anemia -stabilizes Methemoglobin: eliminates O2 binding -cyanosis: blood is blue to chocolate brown

Question 33

Pathologic Mutation in Hb specific (Boston, Black mouth, and Hb Hammersmith)

Answer 33

-Boston: His E7-->Tyr *type of Methemoglobin -Black Mouth: HisF8-->Tyr -Hb Hammersmith: Phe-->Ser *loses heme so it doesn't bind oxygen

Question 34

Pathologic mutation at surface of HB

Answer 34

-HbE: Glu 26 on beta chains -->Lys *not harmful -HbS: Glu 6 on beta chains -->Val *sickle cell anemia *causes precipitation *both have same O2 binding ability *have 4 hemes *iron in Fe2+ state *have same active site

Question 35

sickle cell anemia

Answer 35

-autosomal recessive genetic disorder -can get pain crises -10% african americans are heterozygous for this trait

Question 36

Hbs pathology (valine): causes sickle cell anemia

Answer 36

-E-->V in beta chain decreases surface charge making HbS more hydrophobic -ONLY in T-state (deoxy Hbs) does Val fit into a hydrophobic pocket of beta chain -causes long linear polymers (fibers) to form

Question 37

Hbs pathology in capillaries

Answer 37

HbS loses O2-->deoxy HbS starts to polymerize which causes the blood to sickle *sickled RBCs block capillaries and blood flow

Question 38

Benefit of HbS gene

Answer 38

people who have are heterozygous for HbS gene have a higher survival rate from malaria *25% African Americans

Question 39

difference btwn adult Hb and Fetal Hb

Answer 39

-normal adult Hb (HbA) = alpha2beta2 -Fetal Hb (HbF)= alpha2gamma2 *gamma is a separate gene product made by the fetus

Question 40

O2 delivery to fetus

Answer 40

- in presence of 2,3 BPG, HbF has a higher affinity for O2 than HbA *allows transfer of O2 from the mother (less tightly bound) to the baby (more tightly bonded) -so HbF has a lower affinity for 2,3 BPG than HbA and thus it is less affected by it *gamma chains have Ser instead of His143(+) so they have less affinity for (-) phosphates on 2,3 BPG