Hemoglobin

Question 1

hemoglobin is structurally made of a _____ containing iron

Answer 1

porphyrin

heme = porphyrin + iron

Question 2

why can’t heme travel alone in the blood (why must it be in the larger hemoglobin protein?)

Answer 2

heme itself is very reactive - on its own, it would react another heme and form a non-functional oxidized heme which would aggregate

protein keeps heme away from other hemes (protect heme from auto-oxidation)

*ferrous heme (2+ charge) acts as oxygen carrier, while ferric heme (3+ charge) is nonfunctional

Question 3

heme - structure and function

Answer 3

O2 binding molecule in myoglobin and hemoglobin

porphyrin containing iron (Fe2+)

sequestered in hydrophobic pocket to prevent auto-oxidation (rusting) of iron

Question 4

what does the oxygen dissociation curve look like for myoglobin vs hemoglobin?

Answer 4

myoglobin - hyperbolic (1 binding site)

hemoglobin - sigmoidal (4 cooperative binding sites)

Question 5

what forces hold myoglobin vs hemoglobin together

Answer 5

myoglobin - hydrophobic residues

hemoglobin - inter-subunit salt bridges (and hydrophobic residues holding the inside together)

Question 6

which has a higher affinity for oxygen, hemoglobin or myoglobin? explain why this makes sense

Answer 6

hemoglobin has a LOWER affinity for oxygen (P50 = 25torr) - it makes sense that the oxygen carrier is able to let oxygen go in tissues which have a higher affinity for oxygen

so myoglobin has a higher oxygen affinity (P50 = 1-2 torr) so it can grab it when available

*remember that P50 is the partial pressure of oxygen at which half of the binding sites are saturated

Question 7

structure of adult hemoglobin (HbA)

Answer 7

tetrameric protein - 2 alpha globin and 2 beta globin subunits

a different globin gene encodes each type of subunit

one heme prosthetic group found within hydrophobic pocket in each subunit (4 heme groups total)

Question 8

describe the cooperativity of oxygen binding hemoglobin

Answer 8

first binding site has lowest affinity, but binding causes next heme group to have higher affinity, until the 4th heme group can very easily bind oxygen (affinity is at its highest)

results in sigmoidal oxygen dissociation curve

(due to conformational change of one group from Tense to Relaxed state causing shifting in the others)

*also works in reverse, so it becomes easier to release oxygen in tissues as oxygen dissociates from each heme group

Question 9

a protein that exhibits changes in ligand/substrate affinity under the influence of small molecules

Answer 9

allosteric protein (often multi-subunit)

small molecules bind at sites spatially distant from ligand-binding site —> induce long-range conformational effects

*note that allosteric effectors (the small molecules) can either increase or decrease affinity

Question 10

what are the two conformational states of hemoglobin, and which binds oxygen?

Answer 10

T (tense state) - deoxyhemoglobin

R (relaxed state) - binds oxygen (higher affinity)

Question 11

describe the Bohr effect on hemoglobin and myoglobin

Answer 11

increased [CO2] and acidification of the blood (lower pH) LOWERS the affinity of hemoglobin for oxygen

not true for myoglobin

this is due to pH sensor on Asp and His residues on beta chain (allosteric sites)

[remember that CO2 causes acidification of the blood, and too much CO2 can induce acidosis]

Question 12

describe the mechanism of the Bohr effect in the tense vs relaxed state of hemoglobin

Answer 12

in T state, His and Asp residues in beta chain (pH sensors) are close together

in R state, His and Asp residues are separated and His releases a proton, which lowers the pKa of His

pKa of His is now close to blood (7.1), so slight acidification of the blood can protonated His, making it want to interact ionically with Asp —> induces tense state again

Question 13

CO2 + H2O …..

complete this equilibrium

how is this relevant in the lungs?

Answer 13

CO2 + H2O <> H2CO3 <> H+ + HCO3-

remember H2CO3 = carbonic acid

*in lungs, hemoglobin releases H+, reforming carbonic acid —> CO2 is liberated and H2O is left behind

Question 14

how does CO2 get transported directly with hemoglobin?

Answer 14

forms carbamates with amine groups (N-terminus, lysines, arginines) —> CO2 stabilizes deoxy (T) state of hemoglobin

Question 15

effect of Diamox (acetazolamide)? what is it used for?

Answer 15

promotes excretion of bicarbonate in the kidneys —> lowers blood pH —> lowers affinity of hemoglobin for oxygen

used to treat altitude sickness —> hemoglobin more easily gives up oxygen to tissues

Question 16

mild acidosis triggers hyperventilation

explain why

Answer 16

increased [CO2] causes acidosis

you hyperventilate to blow off the carbon dioxide and increase O2 uptake

remember:
CO2 + H2O <> H2CO3 <> H+ + HCO3-
*you want to keep pushing the equation left so you get rid of acid

Question 17

how does nitric oxide (NO) interact with hemoglobin?

Answer 17

NO binds preferentially to R-state hemoglobin directly on the heme iron

release of NO upon deoxygenation of hemoglobin allows NO to bind vascular endothelium receptors —> vasodilation, increased blood flow to hypoxic tissue

Question 18

what is 2,3 BPG and how does it interact with hemoglobin?

Answer 18

2,3 BPG (biphosphoglycerate) is product of glycolysis in erythrocytes

2,3 BPG binds/stabilizes T form of hemoglobin to induce oxygen release in tissues

increases at high altitude to help body adjust to lower oxygen pressure (more oxygen release at tissues)

Question 19

order these from lowest p50 (highest affinity for oxygen) to highest p50 (lowest affinity for oxygen):
fetal HbF
altitude-adjusted HbA
normal HbA

Answer 19

fetal HbF - lowest p50/highest affinity (want to be able to grab oxygen from mother)

normal HbA

altitude-adjusted HbA - highest p50/lowest affinity (due to 2,3 BPG) (want to be able to release oxygen more easily to tissues at lower oxygen pressures)

Question 20

what 3 factors induce tense state of hemoglobin?

Answer 20

1. acidification (low pH)
2. CO2 (remember high [CO2] also acidifies blood)
3. 2,3 BPG (think high altitude)

Question 21

effect of CO on hemoglobin

how can you treat CO poisoning?

Answer 21

carbon monoxide has MUCH higher affinity for hemoglobin than oxygen - occupies oxygen sites

treat with 100% O2 or hyperbaric O2 to try to displace CO

Question 22

diabetics have up to 3-fold higher concentrations of what form of hemoglobin?

Answer 22

HbA1c: glycosylated Hb (by glucose), occurs non-enzymatically

HbA1c levels are representative of blood glucose over 6-8 weeks (measures avg glucose levels over past 3 months - makes it a good test)

note that glycosylation affects electrophoretic mobility of hemoglobin

Question 23

HbS (sickle cell) results from what mutation?

contrast sickle cell anemia to sickle cell trait

Answer 23

HbS due to glutamine (Glu) —> valine (Val) in beta chain

sickle cell anemia: homozygous (AR)

sickle cell trait: heterozygous, usually asymptomatic except in renal medulla

Question 24

when does sickling (in sickle cell anemia) occur, and how can it be treated?

Answer 24

sickling occurs at high concentrations of deoxy HbS (during physical exertion, for example)

to treat: stimulate production of HbF (fetal hemoglobin, which has higher oxygen affinity that HbA)
1. butyrate
2. hydroxyurea

Question 25

what is the effect of butyrate and hydroxyurea?

Answer 25

stimulate production of HbF, for patients with sickle cell anemia

Question 26

HbS heterozygous individuals are protect against most lethal form of ____

Answer 26

malaria - plasmodium infection of RBC triggers sickling, which triggers phagocytes

(if untreated, HbS homozygotes die before adulthood, so don’t get this advantage)

Question 27

a patient with a defect in alpha-globin chain production (alpha thalassemia) has a high content of tetramers of the beta-globin chains (beta4, aka HbH), which tend to precipitate, causing anemia. In solution, beta4 tetramers are poor for oxygen delivery because of their:
a. greater Bohr effect, compared to HbA
b. higher p50 value, compared to HbA
c. hyperbolic oxygen saturation curve
d. increased binding of 2,3BPG, compared to HbA

Answer 27

c. hyperbolic oxygen saturation curve

both alpha and beta subunits needed for allosteric interactions

Question 28

Met hemoglobin contains….

Answer 28

oxidized form of Fe (iron)

Met Hb = methemoglobin, iron oxidation state is 3+ so it is nonfunctional

Question 29

all of these occur when deoxyhemoglobin is converted to oxyhemoglobin except:
a. specific salt bridges between subunits are broken
b. iron atom is pulled into the plane of the heme group
c. pK values of one His side chain and amino terminus are decreased
d. visible region absorption spectrum changes
e. heme iron undergoes an oxidation from Fe2+ to Fe3+

Answer 29

all are true except last -

ferrous (Fe2+) iron is the only form capable of binding oxygen !

Question 30

T/F: nitric oxide (NO) is released from Hb preferentially at sites of active metabolism because a cysteine has a much higher affinity for NO in oxyhemoglobin than in deoxyHb

Answer 30

TRUE

Question 31

how would a mutation in cytochrome b5 affect hemoglobin?

Answer 31

would result in formation of methemoglobin (3+ iron oxidation state, nonfunctional)

cytochrome b5 normally works with an enzyme that keeps reducing MetHb back to ferrous (2+) form

Question 32

describe how conformational changes in Hb change shape of R and T state?

Answer 32

planarity of heme cofactor changes from puckered (T state) to flat (R state) which allows oxygen to form a stable interaction with iron

Question 33

the residues in 2,3BPG binding site are [anionic/cationic]

Answer 33

cationic (lysines), which electrostatically attract 2,3BPG (which is anionic because of phosphate groups)