Lecture: Globular Proteins

Subject: Biochemistry Module # 1 Lecture # 4

Lecturer: Deanne G. Asdala, M.D. FPAPHSPI Date: August 24, 2017
Transcribers: LEE, MANGAHAS, OLBES, OROSA, YUNZAL (CHECKED BY – MOLINA G.; URSUA I.)

OUTLINE I. HEME PROTEINS

I. HEME PROTEINS • Group of specialized proteins that contain a heme
a. Structure of Heme prosthetic group
II. MYOGLOBIN • Role is dependent on 3D structure
III. HEMOGLOBIN • Hemeproteins for binding O2
IV. HEMOGLOBIN VS MYOGLOBIN o Myoglobin (storage)
V. COOPERATIVE BINDING
o Hemoglobin (transport)
VI. ALLOSTERIC EFFECTOR
a. Oxygen Pressure • Heme is responsible for the red color of blood
b. Carbon Dioxide
c. pH
d. Chloride Ion Structure of Heme
e. 2,3-bisphoglycerate
f. Carbon Monoxide
VII. HEMOGLOBIN GENES
VIII. HEMOGLOBINOPATHIES
IX. APPENDIX

LEARNING OBJECTIVES

• Describe the basic structure of myoglobin and

hemoglobin
• Explain how oxygen binds and its effects in
myoglobin and hemoglobin Structure of Heme
• Explain the effect of carbon monoxide on the affinity
of hemoglobin for oxygen
• Explain how allosteric effectors affect the Hg-
O2dissociation curve and alter the affinity of
hemoglobin for oxygen
• Describe how carbon dioxide is transported in blood Structure of Pyrrole
plasma
• Describe the different hemoglobinopathies and how
they affect oxygen binding

REFERENCES (CITE ALL REFERENCES!!!)

• Harper’s Biochemistry (29th ed., 2011)

• Devlin, Thomas (1997). Biochemistry with Clinical
Correlation. Wiley-Liss, Inc. New York. Structure of Protoporphyrin IX
• Smith, C., Allan Marks, Michael Lieberman. (n.d.).
Mark’s Basic Medical Biochemistry. & Heme is a complex of protoporphyrin IX and Ferrous iron
(Fe2+, reduced state)
Legend:
• Protoporhyrin IX is composed of the following:
Remember Trans/Subject o 4 pyrroles linked by α-methylene bridges to form
Lecturer Book
(Exams) Head
a tetrapyrrole ring
G U & ! o Attachments:
§ 4 Methyl
§ 2 Vinyl
§ 2 Propionate
• Ferrous iron is held in the center by the 4 nitrogen of
the pyrroles
• In both myoglobin and hemoglobin, positions are
coordinated to the side chain of a histidine residue of

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the globin molecule, the other position is available to Adult HbA α 2β 2

bind oxygen. hemoglobin
S
Sickle cell HbS α 2β 2
II. MYOGLOBIN
• Present in cardiac and skeletal muscle Minor adult HbA2 α 2δ 2
• Monomeric heme protein Fetal HbF α 2γ 2
• Storage of oxygen in muscle tissues; oxygen carrier
that increases the rate of oxygen transport within the
muscle cell & produced in all tissues but most pronounced in bone
• Can bind only one molecule of oxygen marrow and liver because of the requirements for the
• Has a greater affinity for oxygen compared to incorporation of the hemoglobin and the cytochrome,
hemoglobin which can unload its oxygen. respectively (Devlin, 1997)
• Oxygen affinity is usually high because it’s doesn’t & consists of a tetrapyrrole ring (Protoporphyrin IX) and a
give off oxygen although it is used by the muscles. ferrous iron (Devlin, 1997)
• Structure: & One molecule of Heme requires 8 molecules each of
Glycine and Succinyl CoA (Mark’s Basic Medical
Biochemistry)
! Sites of Heme Biosynthesis:
o RBC (85%)- heme part of hemoglobin is
synthesized in the immature RBCs-
reticulocytes and erythroblasts
§ Hemoglobin
§ Myoglobin (10%); other heme proteins
include catalase, tryptophan,
pyrrolase
o Liver (15%)- heme is the prosthetic group of
P450 (CYP450 system)
o Mitochondrion/cytosol
• Transports oxygen from the lungs to the capillaries of
Figure 1 Tertiary Structue of Myoglobin the tissues
o Polar groups are exposed and nonpolar groups • Can bind 4 oxygen molecules
are at the interior (makes it water soluble) • α and β are homologous and have similar 3D
structures
& Has 8 right-handed α-helices (named from A to H) • Ferrous iron at the center of the heme forms 2 more
terminated by proline or beta bends th th
bonds ( 5 and 6 coordination sites)
th
& Stabilized by ionic bonds or salt bridges o 5 coordination site = occupied by imidazole ring
of histidine
o Contains 1 heme inserted in a hydrophobic cleft th
2+ o 6 coordination site = remains unoccupied
§ Heme has Fe at the center
§ Histidine residues are located above and
2+
below the Fe
• Oxygen are bound directly to the ferrous ion
• Oxymyoglobin = oxygenated myoglobin

III. HEMOGLOBIN
• Effects of oxygen binding:
o Iron aligns with the plane of the heme
o Without oxygen binding to iron (deoxygenated
hemoglobin), the iron atom is located slightly
below the plane of the heme structure.
o One pair of αβ subunits rotates by 15º
o Tertiary structure of the entire subunit changes
o Other subunits will have increased affinity for
oxygen
o Once an oxygen molecule has bonded to one of
the subunits of hemoglobin, eventually the other

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 GLOBULAR PROTEINS

subunits will have an increased tendency to be § Cooperation makes hemoglobin deliver

bonded by oxygen as well. more O2 to tissues

• States of hemoglobin
o Taut (T) state
§ Deoxygenated (Hgb)
§ Has low affinity for oxygen
o Relaxed (R) state
§ Fully oxygenated (HbO2)
§ Has high affinity for oxygen
§ binding of oxygen to Hb causes the rupture
of some of the polar bonds between the
dimers allowing movement
• Salt bridges in the T structure break progressively as
oxygen is added
• Myoglobin has higher affinity for oxygen than
hemoglobin
• There is no allosteric interaction for binding with
myoglobin

Curve shapes of oxygen binding:

Hemoglobin – sigmoidal
Myoglobin - hyperbolic

VI. ALLOSTERIC EFFECTORS

IV. HEMOGLOBIN VS MYOGLOBIN
• Hemoglobin - tetrametic protein of erythrocytes;
interact in a cooperative fashion that enables this
transporter to offload high proportion of bound
oxygen to peripheral tissues
• Myoglobin - monomeric protein of red muscle (ie
skeletal); binds oxygen tightly as a reserved oxygen
deprivation.
Oxygen Pressure
4 𝑂$ + 𝐻𝑏 ⟷ 𝑛𝐻 + 𝐻𝑏 𝑂$ *
+
• H is directly proportional to pH
• pH is directly proportional to oxygen affinity
• low pH allows O2 to be released
• T state is favored at low pH

V. COOPERATIVE BINDING
o Hb gets fully oxygenated in alveoli of lungs
§ 98% of O2 binding sites are occupied
o Oxygen is released in tissues
§ Saturation level reaches 32%
§ Low oxygen tension = Affinity for oxygen is
low Carbon Dioxide
§ Oxygen delivery is at maximum ,-./012, -1345.-67
o 66% of potential oxygen-binding sites contribute 𝐶𝑂$ + 𝐻$ 𝑂 𝐻$ 𝐶𝑂8 𝐻 9 + 𝐻𝐶𝑂8
to oxygen transport • Isohydric transport
• Allosteric effect o bicarbonates diffuses out of the RBC and is
o Ability to bind O2 is affected by: carried in the blood to the lungs
§ pO2 o ~ 80% of CO2 is transported this way
§ pH • Carbaminohemoglobin (carbhemoglobin or
§ pCO2 carbohemoglobin)
§ 2,3-BPG o Form of CO2 transported in the blood as a
o Interaction of effector at one site affects the dissolved gas (carbamates)
binding of O2 to other sites • Formed with amino terminal of the polypeptide chain
o Binding cooperation displays a sigmoidal (as 𝐶𝑂$ + 𝐻𝑏 − 𝑁𝐻8 ⟷ 2𝐻 9 + 𝐻𝑏 − 𝑁𝐻 − 𝐶𝑂𝑂 =
compared to hyperbolic in myoglobin) behavior in
fractional saturation vs. pO2 graph

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 GLOBULAR PROTEINS

• Tissues: the H+ from H2CO3 is buffered by o HbF binds 2,3-DPG less than HbA (HbF cannot
deoxyhemoglobin, which binds one proton for every 2 form salt bridge so it cannot bind 2,3-DPG) –
oxygen molecules released gives the fetus more access to oxygen than the
• Lungs: mother
o the high pO2 allows effective O2 binding by Hb o In high altitudes: there is an increase in the
leading to the T to R state transition and the number of erythrocytes and in their
release of protons concentrations of hemoglobin and 2,3-DPG
-
o protons combine with HCO3 from tissues and • Oxyhemoglobin dissociation
enters RBC o ↑ unloading of O2, ↓affinity for O2
o carbonic anhydrase reaction reverses to form
H2O and CO2
& A byproduct of glycolysis
o CO2 diffuses out of the blood into the alveoli and
CADET faces Right
released on expiration
CO2
• Bohr Effect
Acid/Altitude
o Decrease in blood pH or an increase in blood
DPG (2,3-DPG)
CO2 concentration will result in hemoglobin
Exercise
proteins releasing their loads of oxygen
Temperature
o Decrease in carbon dioxide or increase in pH will
result in hemoglobin picking up more oxygen

Remember sequencing of events for CO2 transport for exam. CO2

going into and out of the cell sequence of events.

pH
• decrease in pH decreases the amount of oxygen
bound by hemoglobin at any oxygen concentration
• Protonated hemoglobin favors the T-form (deoxy)
hemoglobin structure.
(note: pH and pO2 in tissues are lower than that in Carbon monoxide
lungs)
• Binds coordinately to heme iron similar to oxygen
• Binding is 220 times stronger than that of oxygen
Chloride Ion • Carboxyhemoglobin (HbCO)
o hemoglobin bound with carbon monoxide
• Chloride shift
o shifts hemoglobin to R state
o Aka hamburger shift/ hamburger phenomenon
• Oxygen saturation curve shift to the left and
o Exchange of chloride and bicarbonate ions
sigmoidal shape becomes hyperbola
across the membranes of RBC
o Maintains electrical neutrality • Note: 60% HbCO is fatal

2,3-biphosphoglycerate
• Derived from 1,3-BPG by BPG mutase
• Has poor binding affinity for oxyHb due to compacted
channel
• At T state:
o hemoglobin has a cavity capable of binding 2,3-
BPG
o when 2,3-BPG binds to the cavity, it forms salt
bridges that must be broken to enter R state
• Role of 2,3-BPG
o When absent, hemoglobin binds to O2more • Effects:
readily o Carbon monoxide poisoning - asphyxiation
o Increased 2,3-BPG favors conversion from R to o Treated by hyperbaric oxygen therapy
T state (causes shift to the right in the oxygen (facilitates CO dissociation)
binding curve)
o 2,3-BPG pushes into the oxygenated hemoglobin
and pops the oxygen out

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 GLOBULAR PROTEINS

VII. HEMOGLOBIN GENES • HbA: the major hemoglobin in humans

• The α-globin genes are derived from chromosome 16
and the β-globin genes are on chromosome 11
• Hb synthesis begins in the first few weeks of
embryonic development within the yolk sac
• The major hemoglobin at this stage of development
is a tetramer composed of 2 zeta (ζ) chains encoded
within the α cluster and 2 epsilon (ε) chains from the
β cluster
• HbF: normally synthesized only during fetal
development
• HbA2: first appears 12 weeks after birth- a minor
component of normal adult HB
• HbA1c: has glucose residue attached to β- globin
chains – increased amounts in DM
VIII. HEMOGLOBINOPATHIES – MUTATIONS
IN HEME
• Mutant forms of hemoglobin that causes a group
of hereditary diseases
• Mutations in the globin genes
• Types:
o 1. cause qualitative abnormalities (e.g.
Sickle cell Anemia)
o 2. cause quantitative abnormalities (e.g.
the Thalassaemias)

• By 6-8 weeks of gestation the expression of this
version of hemoglobin declines dramatically
coinciding with the change in hemoglobin synthesis
from the yolk sac to the liver
• Within the β-globin cluster there is an additional set
of genes, the fetal β-globin genes identified as the
gamma (γ) genes
• Shortly before birth there is a smooth switch from
fetal γ-globin gene expression to adult β-globin gene
& Almost all over 1100 known genetic mutations affecting
human hemoglobin are both extremely rare and benign,
presenting no clinical abnormalities. When a mutation
does compromise biologic function the condition is called
a hemoglobinopathy. More than 7% of the world’s
population are carriers for hemoglobin disorders.
UNSTABLE HEMOGLOBINS
• Substitution of a proline, which cannot participate in
alpha-helical structures
o Hemoglobin Saki: βLeu 14→ Pro
o Hemoglobin Genova: βLeu 28→ Pro
• Unstable hemoglobins denature and form Heinz
bodies, which damage the RBC membrane

MUTANT HEMOGLOBINS
• Hemoglobin Cowtown: βHis 146 → Leu
o Destabilizes the T conformation,
resulting in increased oxygen affinity
• Fetal hemoglobin is identified as HbF and has a SICKLE CELL ANEMIA
slightly higher affinity for oxygen than does adult
hemoglobin - allowing the fetus to extract oxygen • Caused by a single nucleotide substitution (A to T) of
more efficiently from the maternal circulation glutamate for valine at position 6 on the β-subunit of
hemoglobin
Table 1: Different forms of haemoglobin • The change converts a glutamic acid codon (GAG) to
a valine codon (GTG), generating a hydrophobic
Form Chain Fraction of total
“sticky patch” on the surface of the β subunit of
compostion hemoglobin BOTH oxy and deoxy HbS
• The form of hemoglobin in patients with sickle cell
HbA α2β2 90%
anemia is referred to as HbS
• Mutation reduces the solubility of deoxyhb and allows
HbF α2γ2 <2%
formation of fibrous polymeric filaments of
HbA2 α2δ2 2-5% deoxyhemoglobin that precipitate in the RBC
• Leads to deformation of the RBC making it relatively
HbA1c α2β2-glucose 3-6% inflexible and unable to traverse the capillary beds

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 GLOBULAR PROTEINS

• Upon exposure to oxygen at the lungs, HbS filaments Hemoglobinopathy Mutation

immediately dissolve Sickle Cell Hemoglobin Glu 6 à Val
• Oxygen deprivation (such as high altitudes or Hemoglobin Saki Leu 14 à Pro
strenuous exercise) and dehydration can contribute Hemoglobin Genova Leu 28 à Pro
to slower capillary passage of erythrocytes Hemoglobin Cowtown His à Leu
Hemoglobin C Glu 6 à Lys
Hemoglobin M His F8 à Tyr

• Repeated cycles of oxygenation and deoxygenation
leads to irreversible sickling. The end result is
clogging of the fine capillaries
• Autosomal (non-sex chromosome) recessive
inherited disease,so both parents must be
heterozygous carriers to produce a homozygous
child
• Treatment: Hydroxyurea increases circulating levels
of HbF, decreasing RBC sickling
THALASSEMIA
• Etymology: “Thalassa” (Mediterranean region such
as Greece, Italy and Cyprus) and “aemia” (anemia)
• α and β thalassemia are often inherited in an
autosomal recessive fashion, both parents must be
carriers in order for a child to be affected
• If both parents carry a hemoglobinopathy trait, there
is a 25% chance with each pregnancy for an affected
child

Note from Transhead:

Ang importante talaga dito. Alam mo ang properties ng bawat
amino acid. The reason why nagkakaroon ng abnormal cell shape
ay dahil greater ang hydrophobic interaction nung protein niya
(from negatively charged amino acid to hydrophobic).
Kaya ‘pag subjected ito sa electrophoresis (laboratory
technique), mas mabilis magmigrate ang HbA to the positive
electrode of the gel DAHIL mas negative ang glutamate as
compared to valine.

HEMOGLOBIN C (HbC)
• Mutation at codon 6 is the conversion to a lysine
codon (GAG to AAG) which results in the generation
of HbC
• This mutated form reduces the normal plasticity of
host erythrocytes
• In those who are heterozygous for the mutation,
about 28–44% of total hemoglobin (Hb) is HbC, and
no anemia develops
• In homozygotes, nearly all Hb is in the HbC form,
result in moderate normocytic hemolytic anemia

HEMOGLOBIN M
• Genetic counseling and genetic testing is
• In hemoglobin M, histidine F8 (His F8) has been recommended for families that carry a thalassemia
replaced by tyrosine trait
• The iron of HbM forms a tight ionic complex with the • Apart from bone marrow transplantation, treatment is
3+
phenolate anion of tyrosine that stabilizes the Fe symptomatic
form
& Partial or total absence of one or more α (alpha
thalassemia) or β (beta thalassemia) chains of hemoglobin

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METHEMOGLOBIN & Myoglobin can be detected in plasma following a

• Heme iron is ferric (Fe+3) rather than ferrous (Fe+2)
and can neither bind nor transport oxygen
• May be caused by sulfonamides, nitrates and nitrites,
Hemoglobin M, or decreased activity of the enzyme
methemoglobin reductase
• Can avidly bind cyanide (CN-) ion, so it may be
induced to treat cyanide poisoning
• 1-2% only in normal people
& In HgB M, His F8 has been replaced by tyrosine. The iron
of HbM forms a tight ionic complex with the phenolate
anion ogtyrosine that stabilizes the Fe3+ form.
myocardial infarction, but assay of serum enzyme provides
more sensitive index of myocardial injury.
ANEMIA
• Reduction in the number of red blood cells or
hemoglobin
• Most commonly due to Iron deficiency
& reduction in the number of RBCs in the blood, can reflect
impaire synthesis of hgb or impaired production of
erythrocytes (e.g. folic acid or Vit. B12 deficiency)
& Dx of anemia begins w/ spectroscopic measurement of
blood hgb levels.

Table 2: Methemoglobin is an altered state of hemoglobin in which the

ferrous (Fe++) irons of heme are oxidized to the ferric (Fe+++) state.
The ferric hemes of methemoglobin are unable to bind oxygen. In
addition, the oxygen affinity of any remaining ferrous hemes in the
hemoglobin tetramer is increased. As a result, the oxygen dissociation
curve is "left-shifted. Argyria ingestion of silver causes blue skin.

POLYCYTHEMIA
• An increased concentration of erythrocytes
• It is usually brought about by low oxygen delivery to
peripheral tissues (compensatory mechanism)
• Polycythemia may be present in people living in high
altitudes

CHESAPEAKE
• an abnormal hemoglobin in which leucine is
substituted for arginine in the α chain(92), results in
high oxygen affinity
• Patient develops polycythemia
GLYCOSYLATED HEMOGLOBIN (HBA1C) Table 3: Summary of hemoglobinopathies
• Glucose is attached to the ε -amino group of lysine Hemoglobinopathy Description
residues and the amino terminals of the β chain of Hemoglobin Saki or Formation of Heinz bodies which damages
hemoglobin Geneva RBC
• Valuable diagnostic tool in diabetes management - Cowtown Destabilizes T form which increases O2
reflects the mean blood glucose concentration over affinity causing a left shift
the preceding 6-8 weeks Sickle cell Mutation from acidic amino acid to nonpolar.
Increased hydrophobic interaction deforms
• 4-6% in normal people; less than 7% in controlled
cell causing sausage-like/sickle shaped
diabetes mellitus
appearance
• Increased glycosylation of hemoglobin increases its HbC Decreased RBC plasticity
affinity for oxygen, therefore preventing its release at HbM
3+
Stabilizes Fe form of Hemoglobin
the tissue and inducing a level of hypoxia in extreme Methemoglobin
3+ 2+
Fe rather than Fe in hemoglobin; can
cases neither bind nor transport O2
Glycosylated Increased O2 affinity; causes left shift;
MYOGLOBINURIA Hemoglobin prevents release of O2 from RBC to tissues
• Presence of myoglobin in urine due to massive Anemia Decreased RBC or Hemoglobin
destruction of muscle fibers (e.g. hazing, vehicular Thalassemia Decreased or absence of alpha or beta
subunit in hemoglobin
accidents)
Polycythemia Increased RBC production

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 GLOBULAR PROTEINS

IX. APPENDIX

Transcribed by: LEE, MANGAHAS, OLBES, OROSA, YUNZAL (CHECKED BY MOLINA G., URSUA I.) 8