Investigation of acid-base state of blood and respiratory function of
erythrocytes. Pathological
forms of hemoglobin.
Investigation of blood plasma proteins of
inflammation acute phase, own and indicator enzymes. Non-protein
nitrogenous containing and nitrogen not containing organic components of blood.
residual nitrogen
Blood is a liquid tissue. Suspended in the watery plasma are seven types of cells and cell fragments.
·
red blood cells (RBCs)
or erythrocytes
·
platelets or thrombocytes
·
five kinds of white blood cells
(WBCs) or leukocytes
o
Three kinds of granulocytes
§
neutrophils
§
eosinophils
§
basophils
o
Two kinds of leukocytes without
granules in their cytoplasm
§
lymphocytes
§
monocytes
If
one takes a sample of blood, treats it with an agent to prevent clotting, and
spins it in a centrifuge,
·
the red cells settle to the bottom
·
the white cells settle on top of
them forming the "buffy coat".
The
fraction occupied by the red cells is called the hematocrit. Normally
it is approximately 45%. Values much lower than this are a sign of anemia.
|

|
Biological functions of the blood
The blood is the most specialized fluid tissue which
circulates in vascular system and together with lymph and intercellular space compounds
an internal environment of an organism.
The blood executes such functions:
1. Transport of gases –
oxygen from lungs is carried to tissues and carbon dioxide from tissues to
lungs.
2. Transport of nutrients to all cells of
organism (glucose, amino acids, fatty acids, vitamins, ketone bodies, trace
substances and others). Substances such as urea, uric acid, bilirubin and
creatinine are taken away from the different organs for ultimate excretion.
3. Regulatory or hormonal function – hormones
are secreted in to blood and they are transported by blood to their target
cells.
4. Thermoregulation
function - an exchange of heat between tissues and blood.
5. Osmotic function-
sustains osmotic pressure in vessels.
6. Protective function- by
the phagocytic action of leucocytes and by the actions of antibodies, the blood
provides the most important defense mechanism.
7. Detoxification function
- neutralization of toxic substances which is connected with their
decomposition by the help of blood enzymes.
Blood
performs two major functions:
·
transport through the body of
o
oxygen and carbon dioxide
o
food molecules (glucose, lipids,
amino acids)
o
ions (e.g., Na+, Ca2+,
HCO3−)
o
wastes (e.g., urea)
o
hormones
o
heat
·
defense of the body against
infections and other foreign materials. All the WBCs participate in these
defenses.
The formation of blood cells (cell types and acronyms are
defined below)
All the
various types of blood cells

·
are produced in the bone marrow
(some 1011 of them each day in an adult human!).
·
arise from a single type of cell
called a hematopoietic stem cell — an "adult" multipotent stem cell.
These stem cells
·
are very rare (only about one in
10,000 bone marrow cells);
·
are attached (probably by adherens junctions)
to osteoblasts
lining the inner surface of bone cavities;
·
express a cell-surface protein
designated CD34;
·
produce, by mitosis, two kinds of
progeny:
o
more stem cells (A mouse that has had
all its blood stem cells killed by a lethal dose of radiation can be saved by
the injection of a single living stem cell!).
o
cells that begin to differentiate
along the paths leading to the various kinds of blood cells.
Which
path is taken is regulated by
·
the need for more of that type of
blood cell which is, in turn, controlled by appropriate cytokines
and/or hormones.
Examples:
·
Interleukin-7 (IL-7)
is the major cytokine in stimulating bone marrow stem cells to start down the
path leading to the various lymphocytes
(mostly B cells
and T cells).
·
Erythropoietin (EPO),
produced by the kidneys, enhances the production of red blood cells
(RBCs).
·
Thrombopoietin (TPO),
assisted by Interleukin-11 (IL-11), stimulates the production of megakaryocytes.
Their fragmentation produces platelets.
·
Granulocyte-macrophage
colony-stimulating factor (GM-CSF), as its name
suggests, sends cells down the path leading to both those cell types. In due
course, one path or the other is taken.
o
Under the influence of granulocyte
colony-stimulating factor (G-CSF), they differentiate into neutrophils.
o
Further stimulated by interleukin-5 (IL-5)
they develop into eosinophils.
o
Interleukin-3 (IL-3)
participates in the differentiation of most of the white blood cells but plays a
particularly prominent role in the formation of basophils
(responsible for some allergies).
o
Stimulated by macrophage
colony-stimulating factor (M-CSF) the granulocyte/macrophage
progenitor cells differentiate into monocytes, macrophages,
and dendritic cells
(DCs).
Biological chemistry of blood cells
Two types of blood cells can be distinguished
- white and red blood cells. White blood cells are called leucocytes. Their
quantity in adult is 4-9 x 109/L.
Red blood cells
are called erythrocytes. Their quantity
in peripheral blood is 4,5-5 x 1012/L.
Besides that, there are also thrombocytes or platelets in blood.
White Blood Cells (leukocytes)
Leucocytes
(white blood cells) protect an organism from
microorganisms, viruses and foreign substances, that provides the immune status
of an organism.
·
are much less numerous than red (the
ratio between the two is around 1:700),
·
have nuclei,
·
participate in protecting the body
from infection,
·
consist of lymphocytes and monocytes
with relatively clear cytoplasm, and three types of granulocytes, whose
cytoplasm is filled with granules.
Leucocytes are divided into
two groups: Granulocytes and agranulocytes. Granulocytes consist of
neutrophils, eosinophils and basophils. Agranulocytes consist of monocytes and
lymphocytes.
http://www.youtube.com/watch?v=8ytkFqAMoa8
http://www.youtube.com/watch?v=ce0Xndms1bc
Neutrophils
Neutrophils comprise of
60-70 % from all leucocytes. Their main function is to protect organisms from
microorganisms and viruses. Neutrophils have segmented nucleus, endoplasmic
reticulum (underdeveloped) which does not contain ribosomes, insufficient
amount of mitochondria, well-developed Golgi apparatus and hundreds of
different vesicles which contain peroxidases and hydrolases. Optimum condition
for their activity is acidic pH. There are also small vesicles which contain
alkaline phosphatases, lysozymes, lactopherins and proteins of cationic origin.
Glucose is the main source
of energy for neutrophils. It is directly utilized or converted into glycogen.
90 % of energy is formed in glycolysis, a small amount of glucose is converted
in pentosophosphate pathway. Activation of proteolysis during phagocytosis as
well as reduction of phosphatidic acid and phosphoglycerols are also observed.
The englobement is accompanied by intensifying of a glycolysis and
pentosophosphate pathway. But especially intensity of absorption of oxygen for
neutrophils - so-called flashout of respiration grows. Absorbed oxygen is spent
for formation of its fissile forms that is carried out with participation
enzymes:
1. NADP*Н -OXYDASE catalyzes formation of super oxide
anion
2. An enzyme
NADH- OXYDASE is responsible for formation
of hydrogen peroxide
3. Мyeloperoxydase catalyzes
formation of hypochloric acid from chloride and hydrogen peroxide
Neutrophils
are motile phagocyte cells that play a key role in acute inflammation. When
bacteria enter tissues, a number of phenomena occur that are collectively known
as acute inflammatory response. When neutrophils and other phagocyte cells
engulf bacteria, they exhibit a rapid increase in oxygen consumption known as
the respiratory burst. This phenomenon reflects the rapid utilization of oxygen
(following a lag of 15-60 seconds) and production from it of large amounts of
reactive derivates, such as O2-, H2O2,
OH. and OCl-
(hypochlorite ion). Some of these products are potent microbicidal
agents. The electron transport chain system responsible for the respiratory
burst contains several components, including a flavoprotein NADPH:O2-oxidoreductase
(often called NADPH-oxidase) and a b-type cytochrome.
The most
abundant of the WBCs. This photomicrograph shows a single neutrophil surrounded
by red blood cells.
Neutrophils
squeeze through the capillary walls and into infected tissue where they kill
the invaders (e.g., bacteria) and then engulf the remnants by phagocytosis.
This
is a never-ending task, even in healthy people: Our throat, nasal passages, and
colon harbor vast numbers of bacteria. Most of these are commensals,
and do us no harm. But that is because neutrophils keep them in check.
However,
·
heavy doses of radiation
·
chemotherapy
·
and many other forms of stress
can
reduce the numbers of neutrophils so that formerly harmless bacteria begin to proliferate.
The resulting opportunistic infection can be life-threatening.
http://www.youtube.com/watch?v=EpC6G_DGqkI&feature=related
Some
important enzymes and proteins of neutrophilis.
Myeloperoxidase
(MPO). Catalyzed following reaction:
H2O2
+ X-(halide) + H+®
HOX + H2O (where X- = Cl-, Br-, I-
or SCN-; HOX=hypochlorous acid)
HOCl,
the active ingredient of household liquid bleach, is a powerful oxidant and is
highly microbicidial. When applied to normal tissues, its potential for causing
damage is diminished because it reacts with primary or secondary amines present
in neutrophils and tissues to produce various nitrogen-chlorine (N-Cl)
derivates; these chloramines are also oxidants, although less powerful than
HOCl, and act as microbicidial agents (eg, in sterilizing wounds) without
causing tissue damage. Responsible for the green color of pus.
NADPH-oxidase.
2O2
+ NADPH ®
2O2- + NADP + H+
Key
component of the respiratory burst. Deficiency may be observed in chronic
granulomatous disease.
Lysozyme.
Hydrolyzes
link between N-acetylmuramic acid and N-acetyl-D-glucosamine found in certain
bacterial cell walls. Abundant in macrophages.
Defensins.
Basic
antibiotic peptides of 29-33 amino acids. Apparently kill bacteria by causing
membrane damage.
Lactoferrin.
Iron-binding
protein. May inhibit growth of certain bacteria by binding iron and may be
involved in regulation of proliferation of myeloid cells.
Neutrophils
contain a number of proteinases (elastase, collagenase, gelatinase, cathepsin
G, plasminogen activator) that can hydrolyze elastin, various types of
collagens, and other proteins present in the extracellular matrix. Such
enzymatic action, if allowed to proceed unopposed, can result in serious damage
to tissues. Most of these proteinases are lysosomal enzymes and exist mainly as
inactive precursors in normal neutrophils. Small amounts of these enzymes are
released into normal tissues, with the amounts increasing markedly during
inflammation. The activities of elastase and other proteinases are normally
kept in check by a number of antiproteinases (a1-Antiproteinase,
a2-Macroglobulin,
Secretory leukoproteinase inhibitor, a1-Antichymotrypsin,
Plasminogen activator inhibitor-1, Tissue inhibitor of metalloproteinase)
present in plasma and the extracellular fluid.
Basophiles
Basophiles make up 1-5% of all blood leukocytes. They
are actively formed in the bone marrow
during allergy. Basophiles take part in
the allergic reactions, in the blood coagulation and intravascular
lipolysis. They have the protein synthesis mechanism, which works due to the
biological oxidation energy . They synthesize the mediators of allergic
reactions – histamine and serotonin, which during allergy cause local
inflammation. Heparin, which is formed in the basophiles, prevents the blood
coagulation and activates intravascular lipoprotein lipase, which splits triacylglycerin.
The
number of basophils also increases during infection. Basophils leave the blood
and accumulate at the site of infection or other inflammation. There they
discharge the contents of their granules, releasing a variety of mediators such
as:
·
histamine
·
serotonin
·
prostaglandins
and leukotrienes
which
increase the blood flow to the area and in other ways add to the inflammatory
process. The mediators released by basophils also play an important part in
some allergic responses such as
·
hay fever and
·
an anaphylactic response
to insect stings.
Eosinophiles
They make up 3-6% of all leukocytes. Eosinophiles as
well as neutrophiles defend the cells from microorganisms, they contain
myeloperoxidase, lysosomal hydrolases. About the relations of eosinophiles with
testifies the growth of their amount during the sensitization of organism, i.e.
during bronchial asthma, helminthiasis. They are able to pile and splits
histamine, “to dissolve” thrombus with the participation of plasminogen and
bradykinin-kininase.
Monocytes
They are formed in the bone marrow. They make up 4-8% of all leukocytes.
According to the function they are called macrophages. Tissue macrophages
derive from blood monocytes. Depending on their position they are called: in
the liver – reticuloendotheliocytes, in the lungs - alveolar macrophages, in
the intermediate substance of connective tissue – histocytes etc. Monocytes are
characterized by a wide set of lysosomal
enzymes with the optimum activity in the acidic condition. The major
functions of monocytes and macrophages are endocytosis and phagocytosis.
Lymphocytes
The amount – 20-25%, are formed in the lymphoid tissue
or thymus, play important role in the formation of humoral and cellular
immunity. Lymphocytes have powerful system of synthesis of antibody proteins,
energy is majorily pertained due to glycolysis, rarely – by aerobic way.
http://www.youtube.com/watch?v=cD_uAGPBfQQ&feature=related
There
are several kinds of lymphocytes (although they all look alike under the
microscope), each with different functions to perform . The most common types
of lymphocytes are
·
B lymphocytes
("B cells"). These are responsible for making antibodies.
·
T lymphocytes
("T cells"). There are several subsets of these:
o
inflammatory T cells
that recruit macrophages and neutrophils to the site of infection or other
tissue damage
o
cytotoxic T lymphocytes
(CTLs) that kill virus-infected and, perhaps, tumor cells
o
helper T cells
that enhance the production of antibodies by B cells
Although bone
marrow is the ultimate source of lymphocytes, the lymphocytes that will become
T cells migrate from the bone marrow to the thymus where they mature. Both B cells and T cells
also take up residence in lymph nodes, the spleen and other tissues where they
·
encounter antigens;
·
continue to divide by mitosis;
·
mature into fully functional cells.
Monocytes
Monocytes
leave the blood and become macrophages and dendritic cells.
This
scanning electron micrograph (courtesy of Drs. Jan M. Orenstein and Emma
Shelton) shows a single macrophage surrounded by several lymphocytes.
Macrophages
are large, phagocytic cells that engulf
·
foreign material (antigens) that
enter the body
·
dead and dying cells of the body.
Thrombocytes
(blood platelets)
Platelets
are cell fragments produced from megakaryocytes.
Blood
normally contains 150,000–350,000 per microliter (µl) or cubic millimeter (mm3).
This number is normally maintained by a homeostatic (negative-feedback)
mechanism .
The amount – less than 1%, they play the main role in
the process of hemostasis. They are formed as a result of disintegration of
megakaryocytes in the bone marrow. Their
–life-time is 7-9 days. In spite of the fact that thrombocytes have no nucleus,
they are able to perform practically all functions of the cell, besides DNA
synthesis.
If
this value should drop much below 50,000/µl, there is a danger of uncontrolled
bleeding because of the essential role that platelets have in blood clotting.
Some
causes:
·
certain drugs and herbal remedies;
·
autoimmunity.
When
blood vessels are cut or damaged, the loss of blood from the system must be
stopped before shock
and possible death occur. This is accomplished by solidification of the blood,
a process called coagulation or clotting.
A blood
clot consists of
·
a plug of platelets enmeshed
in a
·
network of insoluble fibrin
molecules.
Red Blood Cells (erythrocytes)
The
most numerous type in the blood.
·
Women average about 4.8 million of
these cells per cubic millimeter (mm3; which is the same as a
microliter [µl]) of blood.
·
Men average about 5.4 x 106
per µl.
·
These values can vary over quite a
range depending on such factors as health and altitude. (Peruvians living at 18,000 feet may
have as many as 8.3 x 106 RBCs per µl.)
RBC
precursors mature in the bone marrow closely attached to a macrophage.
·
They manufacture hemoglobin until it
accounts for some 90% of the dry weight of the cell.
·
The nucleus is squeezed out of the cell
and is ingested by the macrophage.
·
No-longer-needed proteins are
expelled from the cell in vesicles called exosomes.
Human blood contains 25 trillion of erythrocytes.
Their main function – transportation of O2 and CO2 – they
perform due to the fact that they contain 34% of hemoglobin, and per dry cells
mass – 95%. The total amount of
hemoglobin in the blood equals 130-160 g/l. In the process of erythropoesis the
preceding cells decrease their size. Their nuclei at the end of the process are
ruined and pushed out of the cells. 90% of glucose in the erythrocytes is
decomposed in the process of glycolysis and 10% - by pentose-phosphate way.
There are noted congenital defects of enzymes of these metabolic ways of
erythrocytes. During this are usually observed hemolytic anemia and other
structural and functional erythrocytes’ affections.
This
scanning electron micrograph (courtesy of Dr. Marion J. Barnhart) shows the
characteristic biconcave shape of red blood cells.
Thus RBCs are
terminally differentiated; that is, they can never divide. They live about 120
days and then are ingested by phagocytic cells in the liver and spleen. Most of
the iron in their hemoglobin is reclaimed for reuse. The remainder of the heme
portion of the molecule is degraded into bile pigments
and excreted by the liver. Some 3 million RBCs die and are scavenged by the
liver each second.
Red
blood cells are responsible for the transport of oxygen and carbon
dioxide.
Oxygen Transport
In
adult humans the hemoglobin (Hb) molecule
·
consists of four polypeptides:
o
two alpha (α)
chains of 141 amino acids and
o
two beta (β)
chains of 146 amino acids
·
Each of these
is attached the prosthetic group heme.
·
There is one atom of iron at the center
of each heme.
·
One molecule of oxygen can bind to
each heme.
http://www.youtube.com/watch?v=WXOBJEXxNEo&feature=related
The
reaction is reversible.
·
Under the conditions of
lower temperature, higher pH, and increased oxygen pressure in the capillaries
of the lungs, the reaction proceeds to the right. The purple-red deoxygenated
hemoglobin of the venous blood becomes the bright-red oxyhemoglobin of
the arterial blood.
·
Under the conditions of
higher temperature, lower pH, and lower oxygen pressure in the tissues, the
reverse reaction is promoted and oxyhemoglobin gives up its oxygen.
Carbon Dioxide Transport
Carbon
dioxide (CO2) combines with water forming carbonic acid, which
dissociates into a hydrogen ion (H+) and a bicarbonate ions

:
CO2
+ H2O ↔ H2CO3 ↔ H+ +
HCO3−
95%
of the CO2 generated in the tissues is carried in the red blood
cells:
·
It
probably enters (and leaves) the cell by diffusing through transmembrane
channels in the plasma membrane. (One of the proteins that forms the channel is
the D antigen that is the most important factor in the Rh system
of blood groups.)
·
Once
inside, about one-half of the CO2 is directly bound to hemoglobin
(at a site different from the one that binds oxygen).
·
The
rest is converted — following the equation above — by the enzyme carbonic
anhydrase into
o
bicarbonate
ions that diffuse back out into the plasma and
o
hydrogen
ions (H+) that bind to the protein portion of the hemoglobin (thus
having no effect on pH).
Only
about 5% of the CO2 generated in the tissues dissolves directly in
the plasma. (A good thing, too: if all the CO2 we make were carried
this way, the pH of the blood would drop from its normal 7.4 to an
instantly-fatal 4.5!)
When
the red cells reach the lungs, these reactions are reversed and CO2
is released to the air of the alveoli.

Anemia
is a shortage of
·
RBCs
and/or
·
the
amount of hemoglobin in them.
Anemia
has many causes. One of the most common is an inadequate intake of iron
in the diet.
Red
blood cells have surface antigens that differ between people and that create
the so-called blood groups such as the ABO system and the Rh
system.
An
Essay on Hemoglobin Structure and Function:
Figure 1 is a model of human
deoxyhemoglobin. It was created in RasMol version 2.6 by Roger Sayle
using the pdb coordinates from the pdb file 4hhb. The 3D coordinates were
determed from x-ray crystallography by Fermi, G., Perutz, M. F., Shaanan, B.,
Fourme, R.: The crystal structure of human deoxyhaemoglobin at 1.74 A
resolution. J Mol Biol 175 pp. 159 (1984)
Hemoglobin
is the protein that carries oxygen from the lungs to the tissues and carries
carbon dioxide from the tissues back to the lungs. In order to function most
efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich
atmosphere of the lungs and be able to release oxygen rapidly in the relatively
oxygen-poor environment of the tissues. It does this in a most elegant and
intricately coordinated way. The story of hemoglobin is the prototype example
of the relationship between structure and function of a protein molecule.
Hemoglobin
Structure


A
hemoglobin molecule consists of four polypeptide chains: two alpha chains, each
with 141 amino acids and two beta chains, each with 146 amino acids. The
protein portion of each of these chains is called "globin". The a and b globin chains are very similar in
structure. In this case, a and b refer to the two types of globin. Students
often confuse this with the concept of a helix and b sheet secondary
structures. But, in fact, both the a and b globin chains contain primarily a
helix secondary structure with no b sheets.
Figure 2 is a close up view of one
of the heme groups of the human a chain from dexoyhemoglobin. In this
view, the iron is coordinated by a histidine side chain from amino acid 87
(shown in green.)
Each
a or b globin chain folds into 8 a helical segments (A-H) which, in turn, fold
to form globular tertiary structures that look roughly like sub-microscopic
kidney beans. The folded helices form a pocket that holds the working part of
each chain, the heme.
http://www.youtube.com/watch?v=eor6EK_JP40
A heme group is a flat ring molecule containing
carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the
center. Without the iron, the ring is called a porphyrin. In a heme molecule,
the iron is held within the flat plane by four nitrogen ligands from the
porphyrin ring. The iron ion makes a fifth bond to a histidine side chain from
one of the helices that form the heme pocket. This fifth coordination bond is
to histidine 87 in the human a chain and histidine 92 in the human b chain.
Both histidine residues are part of the F helix in each globin chain. t
The
Bohr Effect
The
ability of hemoglobin to release oxygen, is affected by pH, CO2 and
by the differences in the oxygen-rich environment of the lungs and the
oxygen-poor environment of the tissues. The pH in the tissues is considerably
lower (more acidic) than in the lungs. Protons are generated from the reaction
between carbon dioxide and water to form bicarbonate:
CO2
+ H20 -----------------> HCO3- + H+
This
increased acidity serves a twofold purpose. First, protons lower the affinity
of hemoglobin for oxygen, allowing easier release into the tissues. As all four
oxygens are released, hemoglobin binds to two protons. This helps to maintain
equilibrium towards the right side of the equation. This is known as the Bohr
effect, and is vital in the removal of carbon dioxide as waste because CO2
is insoluble in the bloodstream. The bicarbonate ion is much more soluble, and
can thereby be transported back to the lungs after being bound to hemoglobin.
If hemoglobin couldn’t absorb the excess protons, the equilibrium would shift
to the left, and carbon dioxide couldn’t be removed.
In
the lungs, this effect works in the reverse direction. In the presence of the
high oxygen concentration in the lungs, the proton affinity decreases. As
protons are shed, the reaction is driven to the left, and CO2 forms
as an insoluble gas to be expelled from the lungs. The proton poor hemoglobin
now has a greater affinity for oxygen, and the cycle continues.
Haemoglobin or hemoglobin
(frequently abbreviated as Hb or Hgb) is the iron-containing
oxygen-transport
metalloprotein
in the red
blood cells of the blood
in vertebrates
and other animals; in mammals
the protein makes up about 97% of the red cell’s dry content, and around 35% of
the total content including water. Hemoglobin transports oxygen from the lungs
or gills
to the rest of the body, such as to the muscles,
where it releases the oxygen load. Hemoglobin also has a variety of other
gas-transport and effect-modulation duties, which vary from species to species,
and which in invertebrates may be quite diverse.
The
name hemoglobin is the concatenation of heme and globin,
reflecting the fact that each subunit
of hemoglobin is a globular
protein with an embedded heme
(or haem) group; each heme group contains an iron
atom, and this is responsible for the binding of oxygen. The most common type
of hemoglobin in mammals contains four such subunits, each with one heme group.
Mutations
in the genes
for the hemoglobin protein in humans result in a group of hereditary
diseases termed the hemoglobinopathies,
the most common members of which are sickle-cell
disease and thalassemia.
Historically in human medicine, the hemoglobinopathy of sickle-cell
disease was the first disease to be understood in its mechanism of
dysfunction, completely down to the molecular level. However, not all of such
mutations produce disease states, and are formally recognized as hemoglobin
variants (not diseases).[]

Hemoglobin
(Hb) is synthesized in a complex series of steps. The heme
portion is sythesized in both the the mitochondria
and cytosol
of the immature red blood cell, while the globin
protein portions of the molecule are sythesized by ribosomes in the cytosol [3].
Production of Hb continues in the cell throughout its early development from
the proerythroblast
to the reticulocyte
in the bone
marrow. At this point, the nucleus is lost in mammals, but not in birds
and many other species. Even after the loss of the nucleus in mammals, however,
residual ribosomal RNA allows further synthesis of Hb until the reticulocyte
loses its RNA soon after entering the vasculature (this hemoglobin-synthetic
RNA in fact gives the reticulocyte
its reticulated appearance and name).
The
empirical chemical formula of the most common human hemoglobin is C2952H4664N812O832S8Fe4,
but as noted above, hemoglobins vary widely across species, and even (through
common mutations) slightly among subgroups of humans.
In
humans, the hemoglobin molecule
is an assembly of four globular
protein subunits. Each subunit
is composed of a protein
chain tightly associated with a non-protein heme
group. Each protein chain arranges into a set of alpha-helix
structural segments connected together in a globin
fold arrangement, so called because this arrangement is the same folding
motif used in other heme/globin proteins such as myoglobin.[4][5]
This folding pattern contains a pocket which strongly binds the heme group.
A
heme group consists of an iron
(Fe) atom held in a heterocyclic
ring, known as a porphyrin.
The iron atom, which is the site of oxygen binding, bonds with the four nitrogens
in the center of the ring, which all lie in one plane. The iron is also bound
strongly to the globular protein via the imidazole
ring of a histidine
residue below the porphyrin ring. A sixth position can reversibly bind oxygen,
completing the octahedral group of six ligands. Oxygen binds in an "end-on
bent" geometry where one oxygen atom binds Fe and the other protrudes at
an angle. When oxygen is not bound, a very weakly bonded water molecule fills
the site, forming a distorted octahedron.
The
iron atom may either be in the Fe2+ or Fe3+ state, but
ferrihemoglobin (methemoglobin)
(Fe3+) cannot bind oxygen. In binding, oxygen temporarily oxidizes
Fe to (Fe3+), so iron must exist in the +2 oxidation state in order
to bind oxygen. The body reactivates hemoglobin found in the inactive (Fe3+)
state by reducing the iron center.
In
adult humans, the most common hemoglobin type is a tetramer
(which contains 4 subunit proteins) called hemoglobin A, consisting of
two α and two β subunits non-covalently bound, each made of 141 and
146 amino acid residues, respectively. This is denoted as α2β2.
The subunits are structurally similar and about the same size. Each subunit has
a molecular weight of about 17,000 daltons,
for a total molecular
weight of the tetramer of about 68,000 daltons. Hemoglobin A is the
most intensively studied of the hemoglobin molecules.
The
four polypeptide
chains are bound to each other by salt
bridges, hydrogen
bonds, and hydrophobic
interactions. There are two kinds of contacts between the α and
β chains: α1β1 and α1β2.
Oxyhemoglobin is formed during respiration when oxygen
binds to the heme
component of the protein hemoglobin in red blood cells. This process occurs in
the pulmonary capillaries adjacent to the alveoli
of the lungs.
The oxygen then travels through the blood stream to be dropped off at cells
where it is utilized in aerobic glycolysis
and in the production of ATP
by the process of oxidative
phosphorylation. It doesn't however help to counteract a decrease in
blood pH. Ventilation,
or breathing, may reverse this condition by removal of carbon dioxide, thus
causing a shift up in pH.[6]
Deoxyhemoglobin is the form of hemoglobin without the bound oxygen.
The absorption
spectra of oxyhemoglobin and deoxyhemoglobin differ. The oxyhemoglobine
has significantly lower absorption of the 660 nm wavelength
than deoxyhemoglobin, while at 940 nm its absorption is slightly higher.
This difference is used for measurement of the amount of oxygen in patient's blood
by an instrument called pulse
oximeter.


The
oxidation state of iron in hemoglobin is always +2. It does not change when
oxygen binds to the deoxy- form.
Assigning
oxygenated hemoglobin's oxidation state is difficult because oxyhemoglobin is
diamagnetic (no net unpaired electrons), but the low-energy electron
configurations in both oxygen and iron are paramagnetic. Triplet oxygen, the
lowest energy oxygen species, has two unpaired electrons in antibonding π*
molecular orbitals. Iron(II) tends to be in a high-spin configuration where
unpaired electrons exist in eg antibonding orbitals. Iron(III) has an
odd number of electrons and necessarily has unpaired electrons. All of these
molecules are paramagnetic (have unpaired electrons), not diamagnetic, so an
unintuitive distribution of electrons must exist to induce diamagnetism.
The
three logical possibilities are:
1)
Low-spin Fe2+ binds to high-energy singlet oxygen. Both low-spin
iron and singlet oxygen are diamagnetic.
2)
High-spin Fe3+ binds to .O2- (the superoxide
ion) and antiferromagnetism oppositely aligns the two unpaired electrons,
giving diamagnetic properties.
3)
Low-spin Fe4+ binds to O22-. Both are
diamagnetic.
X-ray
photoelectron spectroscopy suggests that iron has an oxidation state of
approximately 3.2 and infrared
stretching frequencies of the O-O bond suggests a bond length fitting
with superoxide. The correct oxidation state of iron is thus the +3 state with
oxygen in the -1 state. The diamagnetism in this configuration arises from the
unpaired electron on superoxide aligning antiferromagnetically in the opposite
direction from the unpaired electron on iron. The second choice being correct
is not surprising because singlet oxygen and large separations of charge are
both unfavorably high-energy states. Iron's shift to a higher oxidation state
decreases the atom's size and allows it into the plane of the porphyrin ring,
pulling on the coordinated histidine residue and initiating the allosteric
changes seen in the globulins. The assignment of oxidation state, however, is
only a formalism so all three models may contribute to some small degree.
Early
postulates by bioinorganic chemists claimed that possibility (1) (above) was
correct and that iron should exist in oxidation state II (indeed iron oxidation
state III as methemoglobin, when not accompanied by superoxide .O2-
to "hold" the oxidation electron, is incapable of binding O2).
The iron chemistry in this model was elegant, but the presence of singlet
oxygen was never explained. It was argued that the binding of an oxygen
molecule placed high-spin iron(II) in an octahedral field of strong-field
ligands; this change in field would increase the crystal
field splitting energy, causing iron's electrons to pair into the
diamagnetic low-spin configuration.

Binding
and release of ligands induces a conformational (structural) change in
hemoglobin. Here, the binding and release of oxygen illustrates the structural
differences between oxy- and deoxyhemoglobin, respectively. Only one of the
four heme groups is shown.
As
discussed above, when oxygen binds to the iron center it causes contraction of
the iron atom, and causes it to move back into the center of the porphyrin ring
plane (see moving diagram). At the same time, the porphyrin ring plane itself
is pushed away from the oxygen and toward the imidizole side chain of the
histidine residue interacting at the other pole of the iron. The interaction
here forces the ring plane sideways toward the outside of the tetramer, and also
induces a strain on the protein helix containing the histidine, as it moves
nearer the iron. This causes a tug on this peptide strand which tends to open
up heme units in the remainder of the molecule, so that there is more room for
oxygen to bind at their heme sites.
In
the tetrameric form of normal adult hemoglobin, the binding of oxygen is thus a
cooperative
process. The binding affinity of hemoglobin for oxygen is increased by the
oxygen saturation of the molecule, with the first oxygens bound influencing the
shape of the binding sites for the next oxygens, in a way favorable for binding.
This positive cooperative binding is achieved through steric
conformational changes of the hemoglobin protein complex as discussed above, i.e.
when one subunit protein in hemoglobin becomes oxygenated, this induces a
conformational or structural change in the whole complex, causing the other
subunits to gain an increased affinity for oxygen. As a consequence, the oxygen
binding curve of hemoglobin is sigmoidal,
or S-shaped, as opposed to the normal hyperbolic
curve associated with noncooperative binding.
Hemoglobin's
oxygen-binding capacity is decreased in the presence of carbon
monoxide because both gases compete for the same binding sites on
hemoglobin, carbon monoxide binding preferentially in place of oxygen. Carbon dioxide
occupies a different binding site on the hemoglobin. Through the enzyme carbonic
anhydrase, carbon dioxide reacts with water to give carbonic
acid, which decomposes into bicarbonate
and protons:
CO2
+ H2O → H2CO3 → HCO3-
+ H+

The
sigmoidal shape of hemoglobin's oxygen-dissociation curve results from cooperative
binding of oxygen to hemoglobin.
Hence
blood with high carbon dioxide levels is also lower in pH
(more acidic).
Hemoglobin can bind protons
and carbon dioxide which causes a conformational change in the protein and
facilitates the release of oxygen. Protons bind at various places along the
protein, and carbon dioxide binds at the alpha-amino
group forming carbamate.
Conversely, when the carbon dioxide levels in the blood decrease (i.e., in the
lung capillaries), carbon dioxide and protons are released from hemoglobin,
increasing the oxygen affinity of the protein. This control of hemoglobin's
affinity for oxygen by the binding and release of carbon dioxide and acid, is
known as the Bohr
effect.

The
binding of oxygen is affected by molecules such as carbon
monoxide (CO) (for example from tobacco
smoking, cars and furnaces). CO competes with oxygen at the heme binding
site. Hemoglobin binding affinity for CO is 200 times greater than its affinity
for oxygen, meaning that small amounts of CO dramatically reduces hemoglobin's
ability to transport oxygen. When hemoglobin combines with CO, it forms a very
bright red compound called carboxyhemoglobin.
When inspired air contains CO levels as low as 0.02%, headache and nausea
occur; if the CO concentration is increased to 0.1%, unconsciousness will
follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked
by CO.
In
similar fashion, hemoglobin also has competitive binding affinity for cyanide
(CN-), sulfur
monoxide (SO), nitrogen
dioxide (NO2), and sulfide (S2-), including hydrogen
sulfide (H2S). All of these bind to iron in heme without
changing its oxidation state, but they nevertheless inhibit oxygen-binding,
causing grave toxicity.
The
iron atom in the heme group must be in the Fe2+ oxidation state to
support oxygen and other gases' binding and transport. Oxidation to Fe3+
state converts hemoglobin into hemiglobin or methemoglobin
(pronounced "MET-hemoglobin"), which cannot bind oxygen. Hemoglobin
in normal red blood cells is protected by a reduction system to keep this from
happening. Nitrogen dioxide and nitrous
oxide are capable of converting a small fraction of hemoglobin to
methemoglobin, however this is not usually of medical importance (nitrogen
dioxide is poisonous by other mechanisms, and nitrous oxide is routinely used
in surgical anesthesia in most people without undue methemoglobin buildup).
In
people acclimated to high altitudes, the concentration of 2,3-bisphosphoglycerate
(2,3-BPG) in the blood is increased, which allows these individuals to deliver
a larger amount of oxygen to tissues under conditions of lower oxygen tension.
This phenomenon, where molecule Y affects the binding of molecule X to a
transport molecule Z, is called a heterotropic allosteric
effect.
A
variant hemoglobin, called fetal
hemoglobin (HbF, α2γ2), is found in the
developing fetus,
and binds oxygen with greater affinity than adult hemoglobin. This means that
the oxygen binding curve for fetal hemoglobin is left-shifted (i.e., a higher
percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in
comparison to that of adult hemoglobin. As a result, fetal blood in the placenta
is able to take oxygen from maternal blood.
Hemoglobin
also carries nitric
oxide in the globin part of the molecule. This improves oxygen delivery
in the periphery and contributes to the control of respiration. NO binds
reversibly to a specific cystein residue in globin; the binding depends on the
state (R or T) of the hemoglobin. The resulting S-nitrosylated hemoglobin
influences various NO-related activities such as the control of vascular
resistance, blood pressure and respiration. NO is released not in the cytoplasm
of erythrocytes but is transported by an anion exchanger called AE1
out of them.[]
When
red
cells reach the end of their life due to aging or defects, they are
broken down, the hemoglobin molecule is broken up and the iron gets recycled.
When the porphyrin ring is broken up, the fragments are normally secreted in
the bile
by the liver.
This process also produces one molecule of carbon
monoxide for every molecule of heme degraded [4]; this is one of the few
natural sources of carbon
monoxide production in the human body, and is responsible for the normal
blood levels of carbon monoxide even in people breathing pure air. The other
major final product of heme degradation is bilirubin.
Increased levels of this chemical are detected in the blood if red cells are
being destroyed more rapidly than usual. Improperly degraded hemoglobin protein
or hemoglobin that has been released from the blood cells too rapidly can clog
small blood vessels, especially the delicate blood filtering vessels of the kidneys,
causing kidney damage
Decrease
of hemoglobin, with or without an absolute decrease of red
blood cells, leads to symptoms of anemia.
Anemia has many different causes, although iron
deficiency and its resultant iron
deficiency anemia are the most common causes in the Western world. As
absence of iron decreases heme
synthesis, red blood cells in iron deficiency anemia are hypochromic (lacking
the red hemoglobin pigment) and microcytic (smaller than normal). Other anemias
are rarer. In hemolysis
(accelerated breakdown of red blood cells), associated jaundice
is caused by the hemoglobin metabolite bilirubin,
and the circulating hemoglobin can cause renal
failure.
Some
mutations in the globin chain are associated with the hemoglobinopathies,
such as sickle-cell
disease and thalassemia.
Other mutations, as discussed at the beginning of the article, are benign and
are referred to merely as hemoglobin
variants.
There
is a group of genetic disorders, known as the porphyrias
that are characterized by errors in metabolic pathways of heme synthesis. King George
III of the United Kingdom was probably the most famous porphyria
sufferer.
To
a small extent, hemoglobin A slowly combines with glucose
at a certain location in the molecule. The resulting molecule is often referred
to as Hb
A1c. As the concentration
of glucose in the blood increases, the percentage of Hb A that turns into Hb
A1c increases. In diabetics
whose glucose usually runs high, the percent Hb A1c also runs high. Because of
the slow rate of Hb A combination with glucose, the Hb A1c percentage is
representative of glucose level in the blood averaged over a longer time (the
half-life of red blood cells, which is typically 50-55 days).
Hemoglobin
levels are amongst the most commonly performed blood
tests, usually as part of a full blood count or complete
blood count. Results are reported in g/L,
g/dL
or mol/L.
For conversion, 1 g/dL is 0.621 mmol/L. If the total hemoglobin concentration
in the blood falls below a set point, this is called anemia.
Normal values for hemoglobin levels are:
·
Women:
12.1 to 15.1 g/dl
·
Men:
13.8 to 17.2 g/dl
·
Children:
11 to 16 g/dl
·
Pregnant
women: 11 to 12 g/dl
Anemias
are further subclassified by the size of the red blood cells, which are the
cells which contain hemoglobin in vertebrates. They can be classified as
microcytic (small sized red blood cells), normocytic (normal sized red blood
cells), or macrocytic (large sized red blood cells). The hemaglobin is the
typical test used for blood
donation. A comparison with the hematocrit
can be made by multiplying the hemaglobin by three. For example, if the
hemaglobin is measured at 17, that compares with a hematocrit of .51
Glucose
levels in blood can vary widely each hour, so one or only a few samples from a
patient analyzed for glucose may not be representative of glucose control in
the long run. For this reason a blood sample may be analyzed for Hb A1c
level, which is more representative of glucose control averaged over a longer
time period (determined by the half-life of the individual's red blood cells,
which is typically 50-55 days). People whose Hb A1c runs 6.0% or
less show good longer-term glucose control. Hb A1c values which are
more than 7.0% are elevated. This test is especially useful for diabetics.[8]
BIOLOGICAL
BUFFERS OF BLOOD
Acid–base
homeostasis is the part of human homeostasis concerning the proper balance
between acids and bases, also called body pH. The body is very sensitive to its
pH level, so strong mechanisms exist to maintain it. Outside the acceptable
range of pH, proteins are denatured and digested, enzymes lose their ability to
function, and death may occur.
A
buffer solution is an aqueous solution consisting of a mixture of a weak acid
and its conjugate base or a weak base and its conjugate acid. Its pH changes
very little when a small amount of strong acid or base is added to it. Buffer
solutions are used as a means of keeping pH at a nearly constant value in a
wide variety of chemical applications.
Many
life forms thrive only in a relatively small pH range so they utilize a buffer
solution to maintain a constant pH. One example of a buffer solution found in
nature is blood. The body's acid–base balance is normally tightly regulated,
keeping the arterial blood pH between 7.38 and 7.42. Several buffering agents
that reversibly bind hydrogen ions and impede any change in pH exist.
Extracellular buffers include bicarbonate and ammonia, whereas proteins and
phosphate act as intracellular buffers. The bicarbonate buffering system is
especially key, as carbon dioxide (CO2) can be shifted through
carbonic acid (H2CO3) to hydrogen ions and bicarbonate
(HCO3-):
Acid–base
imbalances that overcome the buffer system can be compensated in the short term
by changing the rate of ventilation. This alters the concentration of carbon
dioxide in the blood, shifting the above reaction according to Le Chatelier's
principle, which in turn alters the pH.
The
kidneys are slower to compensate, but renal physiology has several powerful
mechanisms to control pH by the excretion of excess acid or base. In response
to acidosis, tubular cells reabsorb more bicarbonate from the tubular fluid,
collecting duct cells secrete more hydrogen and generate more bicarbonate, and
ammoniagenesis leads to increased formation of the NH3 buffer. In responses to alkalosis, the
kidneys may excrete more bicarbonate by decreasing hydrogen ion secretion from
the tubular epithelial cells, and lowering rates of glutamine metabolism and
ammonium excretion.
This
huge buffer capacity has another not immediately obvious implication for how we
think about the severity of an acid-base disorder. You would think that the
magnitude of an acid-base disturbance could be quantified merely by looking at
the change in [H+] - BUT this is not so.
Because
of the large buffering capacity, the actual change in [H+] is so
small it can be ignored in any quantitative assessment, and instead, the magnitude
of a disorder has to be estimated indirectly from the decrease in the total
concentration of the anions involved in the buffering. The buffer anions,
represented as A-, decrease because they combine stoichiometrically
with H+ to produce HA.
A decrease in A- by 1
mmol/l represents a 1,000,000 nano-mol/l amount of H+ that is hidden from view and this is
several orders of magnitude higher than the visible few nanomoles/l change in
[H+] that is visible.) - As noted above in the comments about the
Swan & Pitts experiment, 13,999,994 out of 14,000,000 nano-moles/l of H+ were hidden on buffers and just to
count the 36 that were on view would give a false impression of the magnitude
of the disorder.
The
major buffer system in the ECF is the CO2-bicarbonate buffer system.
This is responsible for about 80% of extracellular buffering. It is the most
important ECF buffer for metabolic acids but it cannot buffer respiratory
acid-base disorders.
The
components are easily measured and are related to each other by the
Henderson-Hasselbalch equation.
Henderson-Hasselbalch
Equation
pH = pK’a + log10 (
[HCO3] / 0.03 x pCO2)
The
pK’a value is dependent on the temperature, [H+] and the ionic
concentration of the solution. It has a value of 6.099 at a temperature of 37C
and a plasma pH of 7.4. At a temperature of 30C and pH of 7.0, it has a value
of 6.148. For practical purposes, a value of 6.1 is generally assumed and
corrections for temperature, pH of plasma and ionic strength are not used
except in precise experimental work.
The
pK'a is derived from the Ka value of the following reaction:
CO2 + H2O <=> H2CO3 <=> H+ + HCO3-
(where
CO2 refers to
dissolved CO2)
The
concentration of carbonic acid is very low compared to the other components so
the above equation is usually simplified to:
CO2 + H2O <=> H+ + HCO3-
By
the Law of Mass Action:
Ka
= [H+] . [HCO3-] / [CO2] . [H20]
<