Biochemistry Exam 5 - Hemoglobin

Front 1

Hemoglobin

Back 1

-oxygen carrier in blood
-tetramers with quaternary structure (oligomer)
-each of the 4 peptide chains is bound to a heme group
-heterotetramer circulating in RBCs

Front 2

Myoglobin

Back 2

-serves as a reserve supply of O2 in muscles
-facilitates movement of oxygen within muscles
-composed of a single polypeptide chain & one heme group
-a monomer in muscle cells

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Heme Group

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-consists of an organic part, protoporphyrin IX, and an iron atom
-porphyrins contain four pyrolle rings linked by four CH groups (methene bridges)

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Protoporphyrin IX

Back 4

-has 4 methyl, 2 vinyl, & 2 propionic acid side chains attached to a tetrapyrolle ring

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Porphyrins

Back 5

-found in nature
-are compounds in which side chains are substituted for the 8 hydrogen atoms at carbons numbered 1-8 in the pyrolle rings

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Iron atom binds to...

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-the four nitrogen atoms (1-4 coordination positions) in the center of the protoporphyrin ring
-iron can form 2 more bonds; one on each side & perpendicular to the heme plane
-these are termed the 5th & 6th coordination positions

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Ferrous

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-in the 2+ oxidation state
-heme
-ferroprototporphyrin --> ferrohemoglobin
-only ferrohemoglobin can bind oxygen

Front 8

Ferric

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-in the 3+ oxidation state
-hemin
-ferriprotoporphyrin --> ferrihemoglobin (aka methemoglobin)
-cannot bind oxygen

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Polypeptide Chain

Back 9

-polypeptide chain for myoglobin --> composed of 153 AA
-hemoglobin --> composed of 2 alpha (or alpha-like) & 2 non-alpha chains
-each polypeptide is the product of a separate gene & is called a globin chain

Front 10

Golbin Produced During Development

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-Adult (HbA1) --> alpha2beta2
-Adult (HbA2) --> alpha2delta2
-Fetus (HbF) --> alpha2gamma2
-Embryo (Hb Gower 1) --> xi2epsilon2
-Embryo (Hb Gower 2) --> alpha2epsilon2
-Embryo (Hb Portland) --> xi2gamma2

-Xi only produce during 2st trimester of prenatal development --> replaced by alpha
-epsilon only produced during first trimester

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Hb Barts-Hydrops Fetalis Syndrome

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-increased levels of Hb Portland are found in cord blood of infants with alpha thalassemia and who have whats called Hb Barts-Hydrops Fetalis Syndrome

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Appearance of Fetal Hemoglobin (alpha2gamma2)

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-coincides with shift in site of erythropoiesis from yolk sac to liver & spleen
-alpha & gamma chain synthesis predominates after first 10-12 weeks of fetal development

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Beta Chain Appearance

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-shows up near time of birth (can be seen as early as 6-8 weeks though)

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Chain synthesis during first 3 months after birth

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-Beta synthesis rapidly increases as gamma synthesis ceases
-by 6 months, beta chain synthesis reaches its max, and gamma goes does to 1%

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Delta Chain Synthesis

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-begins late in 3rd trimester
-gradually increases during 6-12 months after brith until it reaches max adult level of 2.5%

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HbA1c

Back 16

-formed by the post-translational modification of HbA
-levels are significantly elevated in patients with diabetes mellitus whose blood glucose levels are not well-controlled
-formed by non-enzymatic addition of glucose (glycation) to the N-terminal alpha amino group of each beta chain
an Amadori rearrangement forms stable adduct (aldimine --> ketimine)
-measure of long term glucose levels in diabetics

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3-D structure of a single globin polypeptide chain

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-about 75% of the chain is folded in right handed alpha helical conformation
-the inside consists almost entirely of non-polar residues
-the heme group lies within a non-polar crevice within the chain

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Structure of polypeptide chains

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-places heme in an environment where is can carry oxygen reversibly

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Proximal & Distal Histidine

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-There is a proximal and a distal His in the chain of hemoglobin
-Oxygen comes in and binds near the distal His

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Quaternary Structure

Back 20

-hemoglobin is a globular protein
-the 4 polypeptide chains are bound by hydrogen bonds, salt linkages, & hydrophobic bonds
-heme groups are located in crevices near the exterior of the molecule (1 in each subunit)
-little interaction between 2 alpha & 2 beat chains
-hemoglobin transports H+ & CO2 in addition to O2
-binding of these molecules is controlled by allosteric interactions

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Required Physiological Properties of Hemoglobin

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1) Great Solubility (34% or 5mM)
a) spherical shape
b) polar groups on surface of molecule

2) Transport larege quantities of O2 (18-20mL of O2 / 100 mL of blood)

3) Uptake & release of O2 at appropriate partial pressures of O2
a) saturated with O2 in lungs
b) release sufficient amounts of O2 in tissue

4) Good Buffer

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Oxygen Transported in Blood in 2 Ways

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1) as O2 in solution
2) in reversible chemical combination with hemoglobin of erythrocytes (each Hb can carry 4 O2)

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Oxyhemoglobin

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-Hb combined with O2
-iron remains Fe2+
-oxyhemoglobin can reversibly lose O2 to form deoxyhemoglobin (iron remains Fe2+)

Front 24

Oxygen Dissociation Curves

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-hemoglobin --> sigmoidal curve (due to cooperative binding --> binding of one heme facilitate binding of more heme)
-myoglobin --> hyperbolic curve
-

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Affinity for O2

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Cyt Oxidase > Mb > Hb

-characterized by p50
-Myoglobin p50 = 1-5 mm Hg (torrs)
-Hemoglobin p50 = 26 torrs

Front 26

Hemogolbin Saturation

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-at oxygen tension in arterial blood (100 mm Hg), hemoglobin is 95-98% saturated
-in venous circulation where oxygen tension is lower (~40 mm Hg), oxygen dissociates & oxygen is available to cells

Front 27

Hill Coefficient (nH)

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-measure of cooperativity of O2 binding
-nH for hemoglobin is ~2.8
-nH for Mb is ~1.0 (means there is no cooperativity due to single polypeptide chain)

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Increase in hydrogen ion concentration/decrease in pH (effect on hemoglobin O2 affinity)

Back 28

-decreases oxygen affinity of hemoglobin
-has no effect on myoglobin

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Increase in CO2 tension (effect on hemoglobin O2 affinity)

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-shifts the dissociation curve to the right
-this means that when CO2 is present, hemoglobin has a lower affinity for O2
-about 13% of CO2 transported in blood back to the lungs as carbamate

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Bohr Effect

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-linkage between O2, H+, & CO2
-about 0.5 H+ are taken up per O2 released
-in high CO2, high H+ environment, O2 is released
-hemoglobin is an important physiological buffer

Front 31

Carbamino Compound

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~25% of the effect of CO2 on the oxygen dissociation curve is due to the formation of carboamino compounds

Front 32

Organic Phosphates in RBCs particularly 2,3bisphosphoglycerate (BPG) (effect on hemoglobin O2 affinity)

Back 32

-BPG increases at high altitudes
-reduces oxygen affinity of hemoglobin
-BPG binds in the central cavity of deoxyhemoglobin & interacts with 3 positively charged groups on each beta chain
-decreases oxygen affinity because it stabilizes the quaternary structure of deoxyhemoglobin by non-covalently cross-linking beta chains

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Fetal Oxygen Transfer & Hemoglobin

Back 33

-Hemoglobin F (alpha2gamma2) binds BPG less strongly than Hemoglobin A & consequently has a higher affinity for oxygen
-this allows HbF to obtain oxygen at the expense of HbA on the other side of the placenta
-gamma chain has a Ser substituted for His at position 143 --> losing positive charge = less interaction with negatively charged BPG

Front 34

Increasing concentration of chloride ions (effect on hemoglobin O2 affinity)

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-shifts oxygen dissociation curve to the right
-chloride ions may further brace the deoxy-state by forming additional salt cross bridges

Front 35

Increase in temperature (effect on hemoglobin O2 affinity)

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-decreases oxygen affinity & therefore increases p50
-temperature effects O2 binding of both hemoglobin & myoglobin

Front 36

Oxygen Dissociation Effects (summary)

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-CO2, H+, 2,3-BPG, Cl-, & Temperature --> all decrease O2 affinity

Front 37

Allosteric Properties of Hemoglobin

Back 37

-arise from alpha-beta subunit interactions
-alpha chain by itself has a high oxygen affinity & dhyperbolic dissociation curve (also insensitive to pH, CO2, & BPG)
-isolated beta chains for tetramers called hemoglobin H
-separated chains lose cooperativity effects

Front 38

BPG

Back 38

-net charge --> minus 4-5 charge at physiologic pH
-byproduct of glycolysis
-one binding site on Hb tetramer (in center cavity)

Front 39

[H+], CO2, & BPG

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-functionally competitive
-shift O2 dissociation curve to right

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Oxy/Deoxy Quaternary Structure

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-most of the movement occurs at alpha1beta2(alpha2beta1) interfaces
-minimal movement at a1b1(a2b2) interfaces
-a1b1 shifts 15% relative to a2b2 going from T state (deoxy) to R state (oxy)

Front 41

Salt linkage constraints between chains of Deoxy Hb

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-these constraints are disrupted upon oxygenation

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In order for Hb to become oxygenated...

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-salt linkages must be broken & H+, CO2, and BPG bound to molecule are released

Front 43

When oxygen binds to iron atom...

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-iron moves into plane of Heme
-when there is no O2 bound, the atomic diameter of iron is too large for iron to sit flush with the porphyrin ring
-the iron is displaced toward the proximal His --> moves back into heme plane when O2 is bound

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OxyHb (R-state)

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-salt bridges between subunits are broken
-BPG expelled
-tertiary & quaternary structure changed

Front 45

Cooperation (salt bridges)

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-salt bridges must be broken to bind O2
-easier to break 2nd, 3rd, & 4th salt linkages once 1st one (and consecutive ones) are broken

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Displacement of oxyhemoglobin dissociation curve in various clinical disorders

Back 46

-corrected to pH 7.4

Front 47

Dissociation curve shifted to the right

Back 47

1) Increase in red blood cell 2,3-BPG
-high altitude adaptation
-pulmonary hypoxemia
-cardiac right to left shunt
-**severe anemia; decrease in RBC mass
-congestive heart failure
-decompensated hepatic cirrhosis
-thyrotoxicosis
-hyperphosphatemia (ATP also increased)
2) Functionally abnormal hemoglobin variants

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Dissociation curve shifted to the left

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1) Decrease in red blood cell 2,3-BPG
-septic shock
-severe acidosis
-following transfusion of stored blood
-hypophosphatemia
-panhypopituitarism
-neonatal respiratory distress syndrome
2) Functionally abnormal hemoglobin variants
3) Methemoglobinemia
4) Carbon Monoxide Intoxication

Front 49

Severe Anemia; decrease in RBC mass

Back 49

-compensate for decreased RBCs by increasing [2,3-BPG]

Front 50

Carbomonoxyhemoglobin

Back 50

-hemoglobin combines ~200 times more strongly with CO than with oxygen
-forms a cherry-red compound called carboxyhemoglobin
-Hb + 4 CO --> Hb(CO)4
-headaches & nausea occur is air contains ~0.02% CO
-unconciousness & death can occur if air contains ~0.1% CO
-smoking can block 20% of sites with CO
-CO shows cooperativity as well
-shifts curve to the left & O2 cannot be release to tissue

Front 51

Hb & Mb reduce affinity for CO by...

Back 51

-by forcing a less preferred bent mode of binding

Front 52

Methemoglobin

Back 52

-form of hemoglobin where iron is in ferric (Fe+++) state
-cannot combine with & transport O2
-in normal individuals ~1.7% of hemoglobin is in the form of methemoglobin
-certain drugs can increase the concentration of methemoglobin

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Familial Methemoglobin

Back 53

-patients with a deficiency in the enzyme methemoglobin reductase
-if methemoglobin exceeds 10% of total Hb, patient will have clinically obvious cyanosis
-if methemoglobin reaches 35%, patient will suffer headaches, weakness, & breathlessness

Front 54

Structural Variants (resulting in essentailly normal amounts of an aberrant globin chain)

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-Single Base Substitution --> substitutes one AA for another (typically due to only one base change in a codon)
-Elongated Globin Chain Variants --> can be caused by base substitution in a termination codon, framshift mutation, or failure of cleavage of initiator Met residue
-Shortened Globin Chains --> involve deletion of one or more intact codons
-Non-homologous crossing over --> produce hybrid globin chains

Front 55

Hemoglobin S

Back 55

-amino acid substitution on surface of molecule (altered exterior)
-mutant form of HbA in which Valine is substituted for glutamic acid at position 6 of beta chain
-produces sickle cell anemia

Front 56

Sickle Cell Anemia (epidemiology)

Back 56

1) American Blacks
-7-10% are heterozygous (HbAS; sickle cell trait)
-0.4% are homozygous (HbSS; sickle cell anemia)
2) Protection from malaria --> infected AS cells --> knobs, stick to endothelial cells, clear
3) Treatment
-hydroxyurea, 5-azacytidine (increase HbF)
-bone marrow transplant
-gene therapy

Front 57

DeoxyHbS

Back 57

-insoluble --> sickle cells

Front 58

Altered Exterior

Back 58

-amino acid substitution on surface of molecule --> rarely causes clinical symtoms
-exception is HbS (sickle cell anemia)

Front 59

Altered Active Site

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-defective subunit cannot bind oxygen due to structural change near heme that affects oxygen binding
-when either distal or proximal His is replaced by Tyr, heme is stabilized in ferric form & cannot bind oxygen
-called HbM --> can only exist in heterozygotes since homozygotes would die
-may be misdiagnosed as congenital heart disease in newborns due to cyanosis

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Altered Secondary/Tertiary Structure

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-amino acid substituion can prevent polypeptide chain from assuming normal 3-D conformation
-resulting hemoglobin is usually unstable
-can result in congenital Heinz body hemolytic anemia

Front 61

Altered Quaternary Structure

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-mutations at subunit interfaces can lead to loss of allosteric properties
-often, these mutations increase oxygen affinity of the molecule

Front 62

Thalassemia Syndromes

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-inherited disorder in which production of a single type of globin chain is either diminished or absent
-alpha & beta used to denote which chain is decreased or absent

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Non-deletion form of B-Thalassemia

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-defects involve single base susbtitution or small deletions or insertions within or immediately upstream of the Beta Globin gene
-affects general aspects of gene functional transcription, RNA processing, & RNA translation

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Deletion Forms of B-Thalassemia

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-deletions of different sizes involving B globin gene cluster

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A-Thalassemia due to deletion

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-deletion of one or more a-globin gene
-more complex & graded than B-thalassemia since there are 4 alpha gene loci

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Non-deletion forms of A-Thalassemia

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-non-deletion forms caused by same type of mutation which cause non-deletion forms of B-Thalassemia (however, these types of mutations very rarely cause A-Thalassemia)

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Examples of A-Thalassemia

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-HbH Disease
-Hb Bart's Hydrops Fetalis

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Pathophysiology of B-Thalassemia

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Review Chapter X (Notes 2) - Pg. 10

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Loss of Iron by Excretion

Back 69

-3.5 grams of Iron in Body
-0.1 mg/day lost in urin
-0.1 mg/day by skin desquamation & sweat
-0.3 mg/day by shedding of intestinal muscosa & biliary excretion
-0.5 mg/day due to normal gastrointestinal bleeding

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Additional Iron Needed For...

Back 70

-Rapid Growth
-Lactation
-Pregnancy
-Menstration

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Dietary Iron

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-absorbed mainly by duodenum & jejunum
-heme iron --> found in meats, poultry, & fish
-non-heme iron --> vegetables, fruit, nuts, grain, etc.

Front 72

Transferrin

Back 72

-serum Fe+++ (hemin) transport protein

Front 73

Transferrin Receptor

Back 73

-glycoprotein that mediates uptake of transferrin
-used for cellular uptake/internalization or iron

Front 74

Ferritin

Back 74

-cellular Fe+++ (hemin) storage protein

Front 75

Hemosiderin

Back 75

-denatured, insoluble ferritin

Front 76

Ferrochelatase

Back 76

-enzyme catalyzes insertion of ferrous iron (Fe++) into porphyrin to form heme

Front 77

Ferritin (properties)

Back 77

-24 subunits
-up to 23% iron by weight
-4500 molecules of iron per molecule of ferritin

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Regulation of Iron Absorption by Intestinal Mucosa

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Depends on:
1) Quantity of Iron in diet
2) Composition of diet
3) Behavior of Mucosa of Duodenum & Upper Jejunum