Hematology Module 3: Red Blood Cells and Hemoglobin, Lecture 1-

Erythrocytes and Hemoglobin

Slide 1: Title slide
The first type of blood cells that we will focus on are the erythrocytes, also called the red blood cells. These are
the cells that transport oxygen in the blood from the lungs to the tissues. These cells also help transport some
of the carbon dioxide from the tissues to the lungs. The main function of erythrocytes, namely oxygen
transport, is carried out by the protein that makes up the majority of the protein composition of the cell,
hemoglobin. We will also be looking at how hemoglobin delivers oxygen, and how many factors affect its
affinity for oxygen. Lastly we will be looking at some variations of hemoglobin, one variation of which is
determined by the combination of chains, and which occurs over the lifetime of an individual. The other
variation is based on which molecule binds to hemoglobin, and whether these variants are functional or not.

Slide 2: Erythrocytes biology

First, we will be looking at the molecular composition of the erythrocyte, mostly of the plasma membrane and
the area beneath it. The chemical composition largely determines the functionality of the cell, whether it is fit
to carry out its principal function over its lifetime of 120 days, or it is not fit, in which case it is destroyed
immediately.
We will be looking at the metabolic processes that keep the cells alive. Mature red blood cells are different
from most cells of the human body, such that they do not possess a nucleus and mitochondria. Nevertheless,
the metabolic processes sustain their functionality in their 120-day lifetime. We will see that most of these
processes ensure that hemoglobin remains functional, as the integrity of hemoglobin affects the health and
functionality of the red blood cell.
Additionally, we will look at the differentiation of the red blood cell from early precursors. We will also look at
what regulates their production from the bone marrow. Lastly, we will look at two processes that underlie red
blood cell destruction.

Slide 3: Membrane composition 1

Just like any eukaryotic cell, the erythrocyte is bounded by a lipid bilayer that is semipermeable. The membrane
bilayer has the characteristic arrangement of phospholipids with the polar heads exposed to the aqueous
environment inside and outside the cell, and the nonpolar tails in the hydrophobic region inside the membrane.
Cholesterol is found in roughly equal proportions with the phospholipids. The type of phospholipids, however,
vary in each layer or leaflet. The outer leaflet has mostly phospatidylcholine or PC and sphingomyelin or SM.
The inner leaflet has mostly phospatidylinositol, or PI, phosphatidylethanolamine, or PE, and phosphatidylserine,
or PS. In healthy cells, this asymmetry is maintained by an enzyme called flippase. In senescent red blood cells,
however, the PS tend to shift to the outer leaflet, and this phospholipid shift marks the red blood for
destruction, or apoptosis.
As in other types of eukaryotic cells, the membrane bilayer also contains proteins that cross the membrane
entirely, namely integral proteins, or proteins that span the membrane partly across, namely peripheral
proteins. The integral proteins include the glycophorins, which determine the antigenic properties of red blood
cells, and which also function as membrane anchors with the membrane skeleton, which abounds beneath the
plasma membrane. Other integral proteins include Band 3, which has many functions, one being an anion
exchange protein that transports chloride ions in exchange for bicarbonate ions, which are important for carbon
dioxide transport and maintaining ionic balance in the cell. Band 3 also binds avidly to oxidized and denatured
hemoglobin, which is no longer functional and which is referred to as Heinz bodies.
It is also important to mention here that some integral proteins are receptors such as the receptor for
erythropoietin and the receptor for the iron carrier protein transferrin.
We will talk about some of the peripheral proteins in the next slide.
Slide 4: Membrane composition 2
The characteristic discoid biconcave shape of the red blood cell is partly attributed to the membrane skeleton
that consists of integral proteins like glycophorin and anchor proteins, which link the plasma membrane to
proteins that constitute the lattice inside the cell. The lattice is mainly composed of dimers of the protein
spectrin, which assemble to form tetramers. The tetramer to dimer ratio at any given time determines the
stability of the cell, with a high proportion of tetramers making the cell more stable. The spectrin tetramers are
attached to the integral membrane proteins band 3 and glycophorins via anchor proteins, in this case ankyrin
and protein 4.2 for band 3, and protein 4.1 for glycophorin. Actin is a ubiquitous cytoskeletal protein in animal
cells, and in the red blood cell, it is also part of the lattice network. You do not need to know the other types of
protein.
These proteins are in dynamic equilibrium of association and dissociation with each other, and this equilibrium is
maintained by calcium ions. In sickle-cell anemia, the cells take on a sickled appearance due to crosslinking and
modifications in the membrane skeletal proteins.
The integrity and dynamic stability of the membrane skeleton also underlies the amazing ability of red blood
cells to squeeze through tight spaces in their journey around the body and their ability to withstand
hemodynamic forces. Unfortunately sometimes they do get deformed or fragmented. When the membrane
anchorage to the skeleton is lost, the red blood cell gives off lipid-rich portions of membrane called
microvesicles, and the damaged red blood cell becomes what is called a spherocyte.

Slide 5: Membrane composition 3

The biconcave discoid shape of the red blood cell is also determined by the lipid composition of the membrane
bilayer. Earlier I mentioned cholesterol as being part of the membrane. The cholesterol in the membrane is
often unesterified. This cholesterol exists in equilibrium with unesterified cholesterol in the blood plasma. In
the presence of the enzyme LCAT, the cholesterol is esterified, and in the esterified form, cholesterol cannot
become part of the red blood cell membrane. In LCAT deficiency, however, there is an accumulation of
unesterified cholesterol in the membrane, involving both leaflets. The term discocyte is a general term for a
normally shaped red blood cell. With LCAT deficiency, the discocyte accumulates disproportionately more
cholesterol in the membrane, and the discocyte loses its biconcave discoid shape and turns into what is called
acanthocyte, and as such, can no longer be functional. In this figure, echinocytes and stomatocytes are
variations of the acanthocyte, depending on whether the cholesterol accumulates disproportionately in the
outer or inner leaflet.

Slide 6: Metabolic pathways

Because you learned about osmosis in biology, you probably can predict what happens when red blood cells are
placed in hypertonic or hypotonic solutions. These cells are able to maintain osmotic balance and avoid
shrinking or swelling due to active transport proteins that pump Na and K against their concentration gradients.
Red blood cells also maintain a small amount of Ca inside, because too much calcium can leading to crosslinking
of cytoskeletal proteins and can lead to irreversible deformation. Ca levels are maintained by actively
transporting Ca out of the cell. Active transport of Both Na/K and Ca depends on ATP.
It is also important that hemoglobin stays in the reduced state, as oxidized hemoglobin is nonfunctional. Thus,
maintaining ionic balance and a functional hemoglobin require that the cell generates ATP to meet these
demands. Unlike most other mammalian cells, the mature red blood cell does not synthesize proteins,
therefore the energy demands are met by a simple set of metabolic pathways that does not include aerobic
cellular respiration, or specifically the Krebs cycle. Nevertheless, it is critical that the cell makes enough ATP to
maintain its function and structural integrity.
This table summarizes the 4 metabolic pathways. Glucose is burned by the cell to generate a modest amount of
ATP through glycolysis. The other 3 pathways can be looked at as diversions from glycolysis. Two of these
diversions, namely the hexose monophosphate shunt and the methemoglobin reductase pathways, are
necessary to maintain hemoglobin in a reduced and functional state. The other diversion, the Rapaport-
Leubering pathway, serves to generate a sufficient amount of the molecule BPG, or bisphosphoglycerate, which
affects the affinity of hemoglobin to oxygen.
As you can see, any defects in the pathway leads to serious consequences in hemoglobin function, which largely
determines red blood cell function, and more importantly, in oxygen supply to the tissues.
Slide 7: Membrane composition 4
About 90% of the glucose is used in glycolysis, which generates 2 ATP molecules from each molecule of glucose.
You can see the ATP produced in the yellow part.
The pink square is where glycolysis and the hexose monophosphate pathway overlap. Instead of glucose being
utilized to make ATP, its derivative, glucose-6-phosphate is being used to convert NADP to NADPH. NADPH is
then used to reduce an oxidized form of glutathione to its reduced form, shown here as GSH. Reduced
glutathione, or GSH, is key to maintaining many important molecules, including hemoglobin, in reduced form.
GSH reduces the sulfhydryl groups, or SH, of hemoglobin and other proteins, such as active transport proteins.
Without GSH, hemoglobin becomes oxidized and turns to what I mentioned earlier as Heinz bodies. These
oxidized, denatured hemoglobin attaches to Band 3 protein in the membrane and is recognized by macrophages
in the spleen, which spell destruction for the red blood cell. Active transport proteins that are oxidized can no
longer function, and the cell membrane becomes leaky. A damaged red blood cell due to leaky membrane
proteins also become amenable to destruction by macrophages.
In the green square, the NADH is used to set off a series of reduction reactions that ensure that hemoglobin also
stays in the reduced state. Unlike GSH that reduces the sulfhydryl groups of hemoglobin, this pathway ensures
that the iron in the heme portion of hemoglobin is in the reduced, or ferrous (Fe++) form. Without this
pathway, the iron is in its oxidized form, or ferric (Fe+++) form, which cannot bind oxygen. Hemoglobin that has
oxidized iron is called methemoglobin, and that is why the pathway is called methemoglobin reductase, after
the enzyme that reduces methemoglobin.
(Rapaport- Leuberging Pathway) Lastly the orange square shows when one of the molecules in glycolysis is
diverted to generate 2,3-bisphosphoglycerate or BPG. When BPG binds to hemoglobin, the affinity of
hemoglobin to oxygen decreases, releasing oxygen to the tissues. Without this pathway, oxygen release is
compromised, which leads to tissue hypoxia, or oxygen deficiency.

Hematology Module 3: Red Blood Cells and Hemoglobin, Lecture 2-

Erythrocytes development and destruction
Slide 1: Title slide
From structure we now move on to the development and destruction of red blood cells. Here we will focus on
how they come about and how they differentiate in the bone marrow, and what happens after they reach the
end of their lifespan. Also we address what happens to the hemoglobin the cells contain whey they get
destroyed.

Slide 2: Erythrocytes
From the hematopoietic stem cell, the first cell type that commits to the red blood cell lineage is the BFU-E, or
burst-forming units-erythroid. This is not morphologically distinguishable, but its nature is known from in vitro
cell culture systems. The cell type that comes after is the CFU-E, or colony-forming units-erythroid, again not
identifiable morphologically, but is known from cell culture. Your book defines each cell type. One difference is
the number of cells they generate in a given time period. BFU-E tend to be more quiescent but have a larger
proliferative potential than CFU-E. Another difference is their sensitivity to early versus late-acting cytokines.
BFU-E are more responsive to the early acting cytokines IL-3 and GM-CSF, while CFU-E are more responsive to
erythropoietin, the renal hormone that drives erythrocyte production. CFU-E have more receptors to
transferrin, the main carrier protein for iron. Note that transferrin is not a cytokine. BFU-E and CFU-E are both
progenitor cells.
The generation of morphologically distinguishable cells, also called maturing cells, sets off a series of
differentiation that we will now look at on the next slide. But before we proceed, familiarize yourself with a few
more terms. The first one is erythroblast. Any morphologically identifiable red blood cell precursor in the bone
marrow that possesses a nucleus is an erythroblast. A normally differentiating erythroblast can also be called a
normoblast. A more advanced erythroblast differentiates into a reticulocyte, which has already pinched off its
nucleus. A reticulocyte then gives rise to a mature red blood cell, or erythrocyte.
Slide 3: Erythrocyte development
There are actually six morphologically defined stages of erythropoiesis. The first 4 stages are characterized by
the presence of a nucleus. The cells tend to decrease in size. Initially the nucleus takes up most of the cell, but
the nucleus-to-cytoplasm ratio decreases with differentiation. The nucleus also has a lacy appearance in the
beginning then turns more compact and darker over time. The cytoplasm is initially bluish, or basophilic, due to
RNA, then becomes pink or salmon color, or acidophilic, due to hemoglobin. The polychromatic normoblast is
the last mitotically activate erythroblast. In other words, from this stage on, the cells enter the Go phase and
are permanently quiescent. Also at this stage, hemoglobin production is at its peak. Overall, the normoblasts
spend up to 7 days in the bone marrow.
At the reticulocyte stage, the nucleus is gone, but there is residual RNA and mitochondria, which stain blue with
supravital stains such as methylene blue, as pointed by the horizontal arrow. They are still busy making
hemoglobin, but significantly less than the normoblasts. Reticulocytes may also contain iron granules, which are
identifiable by the stain Prussian blue. As such, they are referred to as siderocytes, whereas normoblasts with
iron deposits are referred to as sideroblasts. Compared to normoblasts, reticulocytes have a shorter time
period, about 2-3 days, before they differentiate to the next stage, which is the mature red blood cell. Many
reticulocytes leave the bone marrow into the peripheral blood before they mature. In certain conditions or
diseases, more reticulocytes spill out into the blood. Reticulocytes are forced out of the bone marrow earlier
than usual. These are called shift or stress reticulocytes. If you see polychromatophilic erythrocytes on a blood
smear, by using Romanowsky stain for example, these are most likely reticulocytes. In certain anemias that
cause a spillover of reticulocytes into circulation, the condition is known as polychromasia.

Slide 4: Erythropoietin
Under conditions of hypoxia, when tissues badly need oxygen, or in anemia, when there is blood loss, for
example, the production of erythropoietin, or EPO, in the kidney increases. EPO levels in the plasma are stable
as long as hemoglobin levels stay within the normal reference range. When hemoglobin goes below 12 g/dL,
however, EPO levels go up. EPO is the main cytokine driving erythroid maturation and proliferation. As I had
mentioned earlier, reticulocytes and their mature derivatives are mitotically inactive. That is, they cannot
respond to EPO. The liver contributes to some EPO. As you can see in this table, EPO has multiple effects, but
they all underlie erythrocyte production. It also shows here that EPO is set off by most anemic conditions, with
one exception. We will look at this in one of the modules concerning anemia.
Other hormones also have similar effects on erythropoiesis. They are testosterone, thyroid hormones, growth
hormone, and the hormones of the adrenal cortex, such as cortisol. Small surprise that some athletes
administer EPO and testosterone to boost their performance in endurance sports, such as the Tour de France.
Before we leave this section, let me point out a couple of limiting factors for erythropoiesis. One factor is iron
supply. In iron deficiency, the EPO effect can only go so far. However, with hemolytic anemia, enough iron is
reused from destroyed red blood cells. With anemia caused by hemorrhage or blood loss, erythropoiesis is
limited by iron stores, but without these stores, you can rescue the situation only by supplemental iron. Related
to this is the amount of iron-saturated transferrin. If this decreases, then erythropoiesis becomes limited. We
will discuss this in the anemia modules.

Slide 5: Extravascular destruction

Like other terminally differentiated cells in the human body, red blood cells have a limited life span. So, as they
reach a point past their prime, they senesce. Mechanisms that prevent oxidation of hemoglobin, active
transport proteins, and other molecules that are key to the cells structural and functional integrity are
diminished over time. The cell loses its osmotic balance, and protein turnover slows down, causing the cell to
lose its ability to withstand hemodynamic forces and retain its flexibility and strength. The asymmetry of the
lipid bilayer shifts, such that more phosphatidylserine or PS accumulates in the outer leaflet, marking the cell for
apoptosis. Much of the destruction of red blood cells is mediated by macrophages in the spleen. These
macrophages can recognize denatured hemoglobin, or Heinz bodies. Essentially it is a sorting out process when
cells are tested for their pliability when they squeeze through smaller openings in the sinusoids. This is a
hypoxic environment that adds insult to injury. Cells that are damaged and old to begin with do not pass the
test and are destroyed by the macrophages. This is extravascular destruction.
 In the macrophage, the hemoglobin is taken apart, giving rise to heme, iron, and globin. The amino acids in
globin are reused to make more proteins. Iron is stored in macrophages or given off to transferrin to transport
for reuse in erythropoiesis in the bone marrow. Heme is converted to bilirubin in the macrophage, and carried
to the liver by albumin. The bilirubin leaves the liver via the bile duct and is converted to urobilinogen prior to
being expelled into the stool or excreted in urine by way of the bloodstream. Some of the urobilinogen returns
to the liver via the enterohepatic cycle.

Slide 6: Intravascular destruction

Intravascular destruction is a little more complicated, but lets focus on the salient features. Hemoglobin,
normally a tetramer, that is, it is made of 4 polypeptide chains, breaks down into dimers, or 2 polypeptide
chains. The dimers are taken up by haptoglobin and transported to the liver. There, hemoglobin is taken apart
into heme, globin, and iron, as in macrophages in extravascular destruction.
When haptoglobin levels are limiting, however, there are two possible fates for hemoglobin. The dimers can be
excreted by the kidney. Some catabolism occurs in the kidney tubules, and the iron released can be stored as
ferritin and hemosiderin. These iron deposits can be detected in tubular cells in urine by Prussian blue, and their
presences, referred to as hemosiderinuria, indicates recent intravascular destruction or hemolysis. The second
possible fate with limited haptoglobin levels is that hemoglobin is oxidized to methemoglobin. Always think
oxidized when you hear or see the word methemoglobin. The methemoglobin is taken apart to give heme
and globin. The amino acids of the globin part are recycled, while heme is transported to the liver by either
albumin or hemopexin. In the liver, iron from the heme can be stored as ferritin, and heme is converted to
bilirubin, as what happens in extravascular destruction.
What do extravascular and intravascular destruction have in common? The amino acids in globin are reused;
iron is reused or stored; and heme is converted to bilirubin and excreted in bile.

Hematology Module 3: Red Blood Cells and Hemoglobin, Lecture 3-Hemoglobin

Slide 1: Title slide
Now we turn to hemoglobin: the very essence of red blood cell functions. This bread and butter is pretty
much the reason for the existence of the erythrocytes. Once hemoglobin loses its structure and functional
integrity, which can be due to geneticor acquired over time, the red blood cell is doomed for destruction.

Slide 2: Hemoglobin structure & function

So, hemoglobin is the main carrier of hemoglobin in red blood cells. Hemoglobin also makes up the bulk of
the protein composition of the red blood cell. We first look at the structure of hemoglobin, followed by the
types of hemoglobin produced depending on life stages. Next we explore the mechanism of oxygen
transport, and also how carbon dioxide is transported. Lastly we look at nonfunctional hemoglobins based
on oxidation status and on what other molecules or atoms can bind to it.

Slide 3: Structure of hemoglobin and heme

You recall from Biology that proteins have 3, if not 4, levels of structural organization. The primary
structure is defined by the sequence of amino acids in the polypeptide chain. The secondary structure is
generated by hydrogen bonding between amino acids of the same polypeptide chain. There are two types
of secondary structure: one is the alpha helix, as shown here, and the other is the beta pleated sheet, not
shown here. Protein domains that have specific biochemical functions are conferred by secondary
structure. The tertiary structure is created from various interactions between the functional groups of the
amino acids in the same polypeptide chain. These interactions include salt bridges, hydrophobic
interactions, and hydrogen bonds. The tertiary structure gives the protein its 3-dimensional shape. The
next structure, the quaternary, is only possessed by proteins that are made of more than one polypeptide
chain, such as hemoglobin. Antibodies, which are made of 4 subunits, also possess a quaternary structure.
In hemoglobin, the subunits are held together by the same interactions that govern the tertiary structure.
Slide 4: Structure of hemoglobin and heme (cont.)
As you can see in this illustration, and in the other one here, hemoglobin is made of two pairs of the same
subunits, represented by two different colors. In adult hemoglobin, also called hemoglobin A, one pair is
made of alpha-2 chains, and the other pair is made of beta-2 chains.
Hemoglobin has two major components: the protein part, which is called globin, and the prosthetic part,
which is heme. Heme is made of a porphyrin ring chelated with an iron atom. In the reduced form, the iron
is a ferrous ion (Fe2+), but in the oxidized form, it is a ferric ion (Fe3+). Only ferrous ion, the reduced form,
can bind to oxygen. Since hemoglobin has four subunits, a molecule of hemoglobin has four heme groups,
and should be able to transport 4 oxygen molecules.
Heme is synthesized in the mitochondria, but only when iron supplies are adequate. In iron deficiency,
heme synthesis is inhibited. The rate-limiting step in heme synthesis is carried out by the enzyme ALAS2.
This enzyme is inhibited during iron deficiency.

Slide 5: Assembly of hemoglobin

As I had mentioned in the previous slide, adult hemoglobin has two types of subunits, alpha-2 and beta-2.
Each of these is encoded by genes from a different chromosome, but their expression is turned on only
when iron supplies are adequate. The presence of heme actually regulates the expression of globin, which
means that when iron is in short supply, heme is not synthesized in the mitochondria, and therefore globin
is not synthesized either.
Globins, just like other proteins, are synthesized in ribosomes. Here you can see that alpha and beta chains
are synthesized independently because they are encoded by two different genes. The newly synthesized
heme associates with the newly synthesized globin chain, and the alpha and beta globins associate
spontaneously, forming stable alpha-beta dimers. When two dimers associate, they form the hemoglobin
tetramer, or a complete hemoglobin. In this illustration, the tetramer is made of two alpha 2 and two beta 2
chains, making up hemoglobin A2, or adult hemoglobin.

Slide 6: Ontogeny of hemoglobin

The alpha genes are located in chromosome 16, while the beta and gamma genes are located in
chromosome 11. Fetal hemoglobin, or Hb F, is alpha-2 gamma-2 tetramer. This has a higher affinity for
oxygen than adult hemoglobin and is the most common type at birth. You can see that hemoglobin F is
used less after 1 year of age.
You can also see that hemoglobin A is synthesized before birth but is not commonly used until after 1 year
of age. So the decrease in hemoglobin F usage is accompanied by an increase in hemoglobin A usage.
Another type of adult hemoglobin, hemoglobin A2, is an alpha-2 delta-2 tetramer. Hemoglobin A2 has a
slightly higher affinity for oxygen than hemoglobin A, but it is not commonly used after 1 year of age and all
throughout life.
The switch from hemoglobin F to hemoglobin A is (in part) reversible. There are conditions where
individuals increase their usage of hemoglobin F, as you can imagine if they have inherited a defective gene
or allele for adult hemoglobin.

Slide 7: Oxygen-Hemoglobin dissociation curve

You must have seen this figure in an earlier course in Biology or Physiology. This is called the oxygen-
hemoglobin dissociation curve. It shows the relationship between the partial pressure of oxygen and the
percent saturation of hemoglobin by oxygen. Partial pressure of oxygen is the pressure it exerts in a
mixture of gases, and is proportional to its concentration. The S shape, or sigmoid shape, of the curve
reflects the fact that more than one oxygen can bind to hemoglobin, and that binding of oxygen facilitates
binding of the same hemoglobin molecule to other oxygen molecules.
Lets focus on the red curve because this is the dissociation curve under normal conditions. We can look at
this curve starting from the top right position of the graph. Notice that the plateau is quite long before it
starts to descend. This means that over a wide range of partial pressures of oxygen, the percent saturation
of hemoglobin remains high at 90% or greater. This provides an excess capacity for oxygen delivery
especially when oxygen availability becomes low. At partial pressures of oxygen in tissues, which usually
average around 40 mm Hg, more than 50% of hemoglobin is still saturated. So oxygen is released at
physiological partial pressures, but it is not readily given off by hemoglobin.
 When blood gets acidic and there is increased partial pressure of carbon dioxide, as in strenuous exercise,
the curve shifts to the right, meaning that more oxygen is given off by hemoglobin. Likewise, when you
exercise, temperature increases, facilitating oxygen release to tissues that demand oxygen, like skeletal
muscles. In the early part of this module, I mentioned bisphosphoglycerate or 2,3-BPG. When BPG binds to
a different pocket in hemoglobin, it decrease affinity of hemoglobin for oxygen, facilitating oxygen release,
which explains the shift of the curve to the right.
On the other hand, increased alkalinity of the blood and decreased carbon dioxide and temperature makes
the hemoglobin hang on to oxygen more, shifting the curve to the left. Likewise, when 2,3-BPG levels go
down, the affinity of hemoglobin to oxygen is higher.
Another way is to look at the graph which partial pressure of Oxygen is the hemoglobin is 50% saturated
with Oxygen. If curve shifts to the right , you need higher oxygen pressure to keep the hemoglobin
saturated meaning the affinity to oxgen is lower oxygen is ready to leave to the tissue. If left, low oxygen
pressure affinity to oxygen is higher oxygen released is hampered.
Quiz: Fetal hemoglobin has a higher affinity for oxygen, so the oxygen dissociation curve shifts to the __.

Slide 8: Oxygen-Hemoglobin affinity

This table summarizes what was illustrated previously. Under conditions where partial pressure of oxygen
is high, the affinity of hemoglobin to oxygen increases, which was illustrated by the plateau of the
dissociation curve. In tissues that are not actively metabolizing or under conditions of low activity,
hemoglobin hangs on to dear oxygen because there is no reason to release more oxygen than necessary.
However, in strenuous exercise or under conditions of high metabolic activity, tissues demand oxygen, and
oxygen release is facilitated.

Slide 9: Oxygen-Hemoglobin affinity (cont.)

The figure on the right shows the spatial relationship of 2-3 BPG, sometimes referred to as DPG, with the
heme groups carrying oxygen. BPG is negatively charged and is attracted to the predominantly positive
charges in the central cavity of hemoglobin. When BPG fits into the pocket, the affinity of hemoglobin to
oxygen decreases. This is an example of the allosteric effect of BPG. You must have encountered this term
in enzymes. Like BPG, H+ and CO2 also have allosteric effects on hemoglobin.
Interestingly, BPG production goes up when you go to high altitudes. This makes sense because the more
BPG, the more oxygen is released, which makes up for the low oxygen concentration in the air in high
altitudes. This is natures quicker way to meet oxygen demands. In time, erythropoietin production goes
up, and by amping up red blood cell production, will also compensate for the thin air.
When red blood cells reach the tissues, H+ and carbon dioxide bind to hemoglobin, facilitating release of
oxygen. The decrease in hemoglobin affinity for oxygen that is induced by H+ and carbon dioxide is called
the Bohr effect. Hemoglobin that has released its cargo of oxygen is called deoxyhemoglobin.
Deoxyhemoglobin that has a cargo of carbon dioxide is called carbaminohemoglobin. When red blood cells
reach the lungs where the partial pressure of oxygen is high, deoxyhemoglobin or carbaminohemoglobin
picks up the oxygen and releases protons and carbon dioxide.

Slide 10: CO2 transport

The carbon dioxide given off by metabolizing tissues is transported in 3 different ways in the blood. 7% is
dissolved in plasma, while 23% is bound to hemoglobin as carbaminohemoglobin. The majority of carbon
dioxide combines with water to form carbonic acid, and this reaction is mediated by the enzyme carbonic
anyhydrase in the red blood cell. Carbonic acid in turn dissociates into H+ and bicarbonate ions. The H+
bind to hemoglobin at different places and facilitates oxygen release to the tisues. The bicarbonate leaves
the cell in exchange for chloride ions to maintain ionic balance in the cell. This exchange is the chloride
shift, and is mediated by the Band 3 protein, as you may recall, in the plasma membrane.
In the lungs, the high partial pressure of oxygen that we inhale displaces the protons from hemoglobin. The
protons combine with bicarbonate, which have been exchanged with chloride through the process of
chloride shift. The reaction that took place in venous blood or in tissues is reversed, generating carbon
dioxide and water. This is the carbon dioxide that we exhale out. This process underlies most of carbon
dioxide transport in the blood. The more carbon dioxide we exhale, the less acidic the blood becomes as
the excess H+ generated from actively metabolizing tissues is removed through CO2 and H2O.
Slide 11: Abnormal hemoglobins
You recall that the iron in heme has to stay in the reduced, or ferrous state, for it to be able to bind oxygen.
In the presence of oxidants, the iron gets oxidized, or turns into ferric ion, and cannot bind oxygen.
Hemoglobin that has oxidized iron is called methemoglobin. This is the reason for the existence of the
methemoglobin reductase pathway, one of the offshoots of glycolysis. Without this enzyme, hemoglobin
stays oxidized, become denatured, and cannot function. Some of them attach to the plasma membrane
through the integral protein Band 3, forming visible Heinz bodies and marking the red blood cell for
destruction by splenic macrophages. You recall this type of destruction is extravascular.
Carbon monoxide, a toxic but odorless gas, has a higher affinity for hemoglobin and competes for oxygen at
exactly the same site in the heme group. Although not all oxygen is necessarily displaced, carbon monoxide
increases the affinity of hemoglobin for oxygen, shifting the dissociation curve to the left, and impairs
oxygen release to tissues, leading to hypoxia, or worse anoxia, or absence of oxygen. Hemoglobin bound
with carbon monoxide is called carboxyhemoglobin. All of us may have varying levels of
carboxyhemoglobin, depending on our exposure to the gas, especially smoking habits or degree of
secondhand smoking. If levels of carboxyhemoglon remain very high, it can cause tissue injury or death.
People who are exposed to sulfur compounds in polluted air, workplaces, or through drugs can have
sulfhemoglobin, where sulfur atoms combine with heme. Unlike carbon monoxide, sulfur atoms shift the
dissociation curve to the extreme right, compromising oxygen delivery of the molecule. To make matters
worse, sulfhemoglobin can combine with carbon monoxide. Other ways by which hemoglobin can be
damaged is through oxidation of sulfhydryl groups in the globin portion of the molecule. Thus, there are
many possible ways to damage hemoglobin, both reversibly and irreversibly. Fortunately, we have
mechanisms that destroy red blood cells containing nonfunctional hemoglobins. However, if oxygen
delivery is compromised and too many red blood cells are destroyed, hypoxia and anemia occur,
undermining our health.