Download Biochem 5

Blood system
As the primary avenue by which tissues are connected to each other and the surrounding environment, the
blood that circulates throughout our body performs a variety of functions.
Major Functions of Blood
PLASMA
Сlinical scientists employed electrophoresis within a cellulose acetate matrix to analyze the protein
composition of plasma. Using this technique, salt-soluble serum protein fraction separated into five major
components designated albumin and the α1-, α2-, β-, and γ-globulins, respectively.
Technique of cellulose acetate zone electrophoresis. (A) A small amount of serum or other fluid is applied to
a cellulose acetate strip. (B) Electrophoresis in electrolyte buffer is performed. (C) Staining enables separated
bands of protein to be visualized. (D) Densitometer scanning reveals the relative mobilities of albumin, α1globulin, β2-globulin, β-globulin, and γ-globulin.
Roughly 70 to 80% of all plasma proteins are synthesized in the liver. These include albumin, fibrinogen,
transferrin, and most components of the complement and blood coagulation cascades. Two prominent
exceptions are von Willebrand factor, which is synthesized in the vascular endothelium, and the γ-globulins,
which are synthesized in the lymphocytes.
Each Plasma Protein Has a Characteristic Half-Life in the Circulation
The half-life of a plasma protein is the time required for 50% of the molecules present at any given moment
to be degraded or otherwise cleared from the blood. For example, the half-lives of albumin and haptoglobin
in healthy adults are approximately 20 and 5 days, respectively. Under normal circumstances, as older
protein molecules are cleared they are replaced by newly synthesized ones, a process called turnover.
During normal turnover, the total concentration of these proteins will remain constant as the countervailing
processes of synthesis and clearance reach a steady state. In certain diseases, the half-life of a protein may
be markedly altered. For instance, in some gastrointestinal diseases such as regional ileitis (Crohn disease),
considerable amounts of plasma proteins, including albumin, may be lost into the bowel through the
inflamed intestinal mucosa. The half-life of albumin in these subjects may be reduced to as little as 1 day, a
condition referred to as a protein-losing gastroenteropathy.
ALBUMIN
The liver synthesizes approximately 12 g of albumin per day, representing about 25% of total hepatic protein
synthesis and half its secreted protein. About 40% of the body’s albumin circulates in the plasma, where it
accounts for roughly three-fifths of total plasma protein by weight (3.4-4.7 g/dL). The remainder resides in
the extracellular space. Because of its relatively low molecular mass (about 69 kDa) and high concentration,
albumin is thought to contribute 75 to 80% of the osmotic pressure of human plasma. Like most other
secreted proteins, albumin is initially synthesized as a preproprotein. Mature human albumin consists of a
single polypeptide chain, 585 amino acids in length, that is organized into three functional domains. Its
ellipsoidal conformation is stabilized by a total of 17 intrachain disulfide
bonds. A major role of albumin is to bind to and transport numerous ligands. These include free fatty acids
(FFA), calcium, certain steroid hormones, bilirubin, copper, and tryptophan. A variety of drugs, including
sulfonamides, penicillin G, dicumarol, and aspirin, also bind to albumin; a finding with important
pharmacologic implications. Preparations of human albumin have been widely used in the treatment of
burns and of hemorrhagic shock. Some humans suffer from genetic mutations that impair their ability to
synthesize albumin. Individuals whose plasma is completely devoid of albumin are said to exhibit
analbuminemia. Surprisingly, persons suffering from analbuminemia display only moderate edema.
Depressed synthesis of albumin also occurs in a variety of diseases, particularly those of the liver. The plasma
of patients with liver disease often shows a decrease in the ratio of albumin to globulins (decreased albuminglobulin ratio). The synthesis of albumin decreases relatively early in conditions of protein malnutrition, such
as kwashiorkor.
C-reactive protein (CRP, so named because it reacts with the C polysaccharide of pneumococci), α1antiproteinase, haptoglobin, α1-acid glycoprotein, and fibrinogen are classified as acute-phase proteins.
Acute-phase proteins are believed to play a role in the body’s response to inflammation. C-reactive protein
stimulates the complement pathway, while α1-antitrypsin neutralizes certain proteases released during
acute inflammation. The levels of acute-phase proteins may increase by 50% to as much as 1000-fold (in the
case of CRP) during chronic inflammatory states and in patients with cancer. Interleukin 1 (IL-1), a
polypeptide released from mononuclear phagocytic cells, is the principal—but not the sole— stimulator of
acute-phase reactant synthesis by hepatocytes. Additional molecules such as IL-6 also participate. Because
its concentration can rise so dramatically, CRP is used as a biomarker of tissue injury, infection, and
inflammation.
Haptoglobin
During the course of red blood cell turnover, approximately 10% of an erythrocyte’s hemoglobin escapes
into the circulation. This free, extracorpuscular hemoglobin is sufficiently small at ≈65 kDa to pass through
the glomerulus of the kidney into the tubules, where it tends to form damaging precipitates. Haptoglobin
(Hp) is a plasma glycoprotein that binds extracorpuscular hemoglobin (Hb), forming a tight noncovalent
complex (Hb-Hp). Human haptoglobin exists in three polymorphic forms, known as Hp 1-1, Hp 2-1, and Hp 22 that reflect the patterns of inheritance of two genes, designated Hp1 and Hp2. Homozygotes synthesize Hp
1-1 or Hp 1-2, respectively, while Hp 2-1 is synthesized by heterozygotes. Normally, the level of haptoglobin
in a deciliter of human plasma is sufficient to bind 40 to 180 mg of hemoglobin. Since the resulting Hb-Hp
complex is too large (≥ 155 kDa) to pass through the glomerulus, the kidney is protected from the formation
of harmful precipitates while the loss of the iron associated with extracorpuscular hemoglobin is reduced.
Certain other plasma proteins bind heme, but not hemoglobin. They include a β1-globulin hemopexin, which
binds free heme, and albumin, which binds metheme (ferric heme) to form methemalbumin.
Methemalbumin subsequently transfers this metheme to hemopexin.
Haptoglobin Can Serve as a Diagnostic Indicator
In situations where hemoglobin is constantly being released from red blood cells, such as occurs in hemolytic
anemias, the level of haptoglobin can fall dramatically. This decrease reflects the marked difference in the
half-lives of free haptoglobin, approximately 5 days, and the Hb-Hp complex, approximately 90 minutes. The
level of haptoglobin-related protein, a homologue of haptoglobin also present in plasma, is elevated in some
patients with cancers, although the significance of this is not understood.
α1-Antiproteinase
A 394-residue glycoprotein that makes up >90% of the α1-albumin fraction, is the principal serine protease
inhibitor (serpin) in human plasma. Formerly called α1-antitrypsin, α1-antiproteinase inhibitstrypsin,
elastase, and other serine proteases by forming an inactivecovalent complex with them. α1-Antiproteinase is
synthesized by hepatocytes and macrophages. At least 75 polymorphic forms of this serpin, or Pi, exist. The
major genotype is MM, whose phenotypic product is PiM. A deficiency in α1-antiproteinase plays a role in
some cases (~ 5%) of emphysema, particularly in subjects with the ZZ genotype (who synthesize PiZ) and in
PiSZ heterozygotes, both of whom secrete lower levels of serpins than PiMM individuals.
Oxidation of Met358 Inactivates α1-Antiproteinase
In the lungs, components of the smoke produced by burning tobacco products and industrial activities can
oxidize a key methionine residue, Met358, located in the protease-binding domain of α1-antiproteinase.
Oxidation renders α 1 -antiproteinase unable to covalently bind and neutralize serine proteases. The
subsequent damage produced by unchecked proteolytic activity in the lungs can contribute to the
development of emphysema. Smoking can be particularly devastating for patients who already have low
levels of α1-antiproteinase (eg, PiZZ phenotype). Intravenous administration of serpins (augmentation
therapy) has been used as an adjunct in the treatment of patients with emphysema that exhibit α1antiproteinase deficiency. Individuals deficient in α1-antiproteinase are also at greater risk of lung damage
from pneumonia or other conditions that induce the accumulation of polymorphonuclear white blood cells
in the lung. Deficiency of α1-antiproteinase is also implicated in α1-antitrypsin deficiency liver disease, a
form of cirrhosis that afflicts persons possessing the ZZ phenotype. In these individuals, substitution of
Glu342 by lysine promotes the formation of polymeric aggregates of α1-antiproteinase in the cisternae of
the endoplasmic reticulum in hepatic cells.
α2-Macroglobulin
A member of the thioester plasma protein family, comprises 8 to 10% of the total plasma protein in humans.
This homotetrameric glycoprotein is the most abundant member of a group of homologous plasma proteins
that include complement proteins C3 and C4. α2-Macroglobulin is synthesized by monocytes, hepatocytes,
and astrocytes.
DEPOSITION OF PLASMA PROTEINS IN TISSUES LEADS TO AMYLOIDOSIS
Amyloidosis refers to an impairment of tissue function that results from the accumulation of insoluble
aggregates of proteins in the interstitial spaces between cells. The term is a misnomer, as it was originally
thought that the fibrils were starch-like in nature. The fibrils generally are made up of proteolytic fragments
of plasma proteins whose conformation is rich in β-pleated sheet. They generally also contain a P
component derived from a plasma protein closely related to C-reactive protein called serum amyloid P
component. Structural abnormalities or overproduction of more than 20 different proteins have been
implicated in various types of amyloidosis. Primary amyloidosis typically is caused by a monoclonal plasma
cell disorder that leads to the accumulation of protein fragments derived from immunoglobulin light chains.
Secondary amyloidosis results from an accumulation of fragments of serum amyloid A (SAA) consequent to
chronic infections or cancer. In these instances, elevated levels of inflammatory cytokines stimulate the liver
to synthesize SAA, which leads to a concomitant rise in its proteolytic degradation products. Familial
amyloidosis results from accumulation of mutated forms of certain plasma proteins such as transthyretin.
Over 80 mutationally altered forms of this protein have been identified. Patients undergoing regular, longterm dialysis are at risk from β2-microglobulin, a plasma protein that is retained by dialysis membranes.
PLASMA IMMUNOGLOBULINS DEFEND AGAINST INVADERS
The three major components of the body’s immune system are B lymphocytes (B cells), T lymphocytes (T
cells), and the innate immune system. B lymphocytes are mainly derived from bone marrow cells, while T
lymphocytes originate from the thymus. B cells are responsible for the synthesis of circulating, humoral
antibodies, also known as immunoglobulins. The T cells are involved in a variety of important cell-mediated
immunologic processes such as graft rejection, hypersensitivity reactions, and defense against malignant
cells and many viruses. B and T cells respond in an adaptive manner, developing a targeted response for
each invader encountered. The innate immune system defends against infection in a nonspecific manner.
Immunoglobulins
Immunoglobulins are oligomeric glycoproteins whose individual subunits traditionally have been classified as
heavy (H) or light (L) based on their migration during SDS-polyacrylamide gel electrophoresis. Human
immunoglobulins can be grouped into five classes abbreviated as IgA, IgD, IgE, IgG, and IgM.
The most abundant of the five, IgG, consists of two identical light chains (23 kDa) and two identical heavy
chains (53-75 kDa) linked together by a network of disulfide bonds. The type of H chain determines the class
of immunoglobulin and thus its effector function (see below): α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ(IgM). The
γ chains of IgG are organized into four conserved domains: an amino-terminal variable region (VH) and three
constant regions (CH1,CH2, CH3). The five types of H chains are distinguished by differences in their CH
regions. The μ and ε chains each have four CH domains rather than the usual three.
Structure of IgG.
The molecule consists of two light (L) chains and two heavy (H) chains. Each light chain consists of a variable
(VL) and a constant (CL) region. Each heavy chain consists of a variable region (VH) and a constant region
that is divided into three domains (CH1,CH2, and CH3). The CH2 domain contains the complement-binding
site and the CH3 domain contains a site that attaches to receptors on neutrophils and macrophages. The
antigen-binding site is formed by the hypervariable regions of both the light and heavy chains, which are
located in the variable regions of these chains. The light and heavy chains are linked by disulfide bonds, and
the heavy chains are also linked to each other by disulfide bonds. The IgG light chain can be divided into a Cterminal constant region (CL) and amino-terminal variable region (VL). There are two general types of light
chains, kappa (κ) and lambda (λ), which can be identified by their distinct CL regions. In human
immunoglobulins, κ chains are more common than λ chains. A given immunoglobulin molecule always
contains either two κ or two λ light chains—never a mixture of a κ and a λ. IgG molecules are divalent. The
tip of each Y contains an antigen-binding site made up of VH and VL domains that form two antiparallel
sheets of amino acids. The site on the antigen to which an antibody binds is termed an antigenic
determinant, or epitope. Because the region between the CH1 and CH2 domains can be readily cleaved by
pepsin or papain, it is referred to as the hinge region. The hinge region confers flexibility to the Fab arms,
which facilitates binding to epitopes that may be far apart or on two separate antigens. By linking antigen
particles together, large antibody-antigen clusters form that are readily recognized for disposal by
phagocytic leukocytes. Cluster formation is often demonstrated in the laboratory by the formation of
erythrocyte rosettes.
Some immunoglobulins such as IgG exist only as the basic tetramer. Others such as IgA and IgM can form
higher oligomers comprised of two, three (IgA), or five (IgM) copies of the core tetrameric unit.
Variable Regions Confer Binding Specificity
The variable regions of the immunoglobulin light and heavy chains form the antigen-binding sites that confer
antibodies with their amazing specificity. Within the variable regions of the L and H chains are a handful of
hypervariable regions, short (5-10 residue) islands interspersed within the relatively invariable framework or
complementarity-determining regions (CDRs). An antigen-binding site is formed when the hypervariable
regions of an H and L chain align together to form a projecting loop from the antibody surface. As their name
implies, no two variable regions in the immunoglobulins from different individuals share the same amino
acid sequence.
THE COMPLEMENT SYSTEM
The plasma-borne arm of the innate immune system is called the complement system, which can be
activated by antibody-antigen complexes and thus acts consequent to and in support of or as a complement
to the adaptive immune system. The complement system displays features reminiscent of the blood’s
coagulation cascade. Both consist of sets of circulating zymogens (proproteins) that remain catalytically
dormant until activated by proteolytic cleavage. These proteins, called complement factors, are synthesized
by a variety of cell types, including hepatocytes, macrophages, monocytes, and intestinal endothelial cells.
As is the case for clotting factors, most complement factors are proproteases that, upon activation, target
other components of the system, thereby generating a series or cascade of proteolytic activation events that
amplify the production of the system’s protective end products.
Membrane attack complex (MAC).
MAC kills bacterial invaders by binding to and opening a pore in their plasma membrane. Following lysis, the
bacterial remains are destroyed by phagocytic macrophages. Meanwhile, the C3a and C5a proteins serve as
chemoattractants that recruit leukocytes to the site of infection and stimulate an inflammatory response.
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Most plasma are synthesized in the liver. The majority are glycosylated.
Albumin accounts for roughly 60%, by mass, of the protein content of plasma. As such, it is the
principal determinant of intravascular osmotic pressure. Albumin also binds to and transports fatty
acids, bilirubin, metal ions, and certain drugs.
Haptoglobin binds extracorpuscular hemoglobin to prevent its escape into the kidney, preventing
the formation of damaging precipitates in the tubules.
α1-Antitrypsin is the major serine protease inhibitor of plasma.Genetically produced deficiencies of
this protein can lead to emphysema and liver disease.
α2-Macroglobulin is a major plasma protein that neutralizes many proteases and targets select
cytokines to specific organs.
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Humans can synthesize immunoglobulins that specifically target millions of different antigens. The
core structure of the immunoglobulins is a tetramer consisting of two light and two heavy chains
arranged in a “Y” configuration.
Synthesis of diverse antibodies from a limited set of genes is made possible by combining,
rearranging, and somatic mutation of immunoglobulin genes.
The ability to synthesize antibodies against novel antigens represents the defining feature of the
adaptive immune system.
The complement system is generally activated by complexes formed between infecting microbes
and protective antibodies or between mannose-rich polysaccharides on the pathogen’s surface
andmannose-binding protein.
The complement system is activated by a series of proteolytic cleavage events that transform
dormant zymogens into active proteases.
Red Blood Cells
The packaging of hemoglobin and carbonic anhydrase inside specialized cells called erythrocytes greatly
amplified the capacity of circulating blood to carry oxygen to and carbon dioxide away from peripheral
tissues. Anemia, a deficiency in the level of circulating hemoglobin (< 120-130 g/L), compromises health by
reducing the ability of the blood to supply tissues with adequate levels of oxygen. Anemia can arise from a
variety of causes that include genetic abnormalities (eg, sickle cell trait, pernicious anemia), excessive
bleeding, insufficiencies of dietary iron or vitamin B12 or the lysis of red blood cells by invading pathogens
(eg, malaria).
The structure and composition of red blood cells reflect their highly specialized function: to deliver the
maximum quantity of oxygen to tissues and aid in the removal of carbon dioxide, a waste product of cellular
respiration, and urea. The interior of a red blood cell contains a massive
amount of hemoglobin, roughly one-third by weight (30-34 g/dL for an adult). This extraordinary hemoglobin
concentration is achieved, in part, by dispensing with the intracellular organelles normally found in
eukaryotic cells (eg, nucleus, lysosome, Golgi apparatus, mitochondria). As a consequence, mature
enucleated red blood cells are unable to reproduce. Red blood cells possess an extensive cytoskeletal
network responsible for maintaining their biconcave configuration.
Erythrocytes Generate ATP Exclusively via Glycolysis
Mature red blood cells lack the mitochondria that contain ATP synthase and the enzymes of the tricarboxylic
acid cycle (TCA), electron transport chain, and β-oxidation pathway. They are therefore incapable of utilizing
fatty acids or ketone bodies as metabolic fuel. Consequently, red blood cells are completely reliant of
glycolysis to generate ATP. Glucose enters red blood cells by facilitated diffusion, a process mediated by
glucose transporter 1 (GLUT1), also known as glucose permease.
Carbonic Anhydrase Facilitates CO2 Transport
Like oxygen, the solubility of carbon dioxide in aqueous solution is low, much too low to accommodate more
than a few percent of the CO2 produced by metabolically active tissues. However, the solubility of the
hydrated form of CO2, carbonic acid (H2CO3) and its protonic dissociation product, bicarbonate (HCO3−) is
relatively high. The presence in erythrocytes of high levels of the enzyme carbonic anhydrase enables them
both to absorb waste CO2 by catalyzing its rapid conversion to carbonic acid, and to reverse this process in
order to facilitate its expulsion in the lungs. While red blood cells carry some CO2 in the form of hemoglobinbound carbamates, roughly 80% is carried as dissolved carbonic acid and bicarbonate.
About Two Million Red Blood Cells Enter the Circulation per Second
The 120-day lifespan of a normal red blood cell requires that nearly 1% of the 20 to 30 trillion erythrocytes in
a typical individual must be replaced daily. This equates to a rate of production of ~ 2 million new red blood
cells per second. When initially formed, differentiated red blood cells retain a portion of the ribosomes,
endoplasmic reticulum, mitochondria, etc that were present in their nucleated precursors. Consequently,
during the ≈ 24 hours required to complete maturation to erythrocytes, these nascent red blood cells, called
reticulocytes, retain the capacity to synthesize polypeptides (eg, globin) under the direction of vestigial
mRNA molecules.
Erythropoietin Regulates Production of Red Blood Cells
The initial stages of erythropoiesis, the production of red blood cells, are modulated by stem cell factor,
colony-stimulating factors, and interleukins 1, 3, and 6. Commitment of myeloid progenitor cells to
differentiation into erythrocytes is largely dependent on erythropoietin (EPO), a glycoprotein of 166 amino
acids (molecular mass ≈ 34 kDa). EPO, which is synthesized mainly by the kidney, is released into the
bloodstream in response to hypoxia. Upon reaching the bone marrow, EPO stimulates red blood cell
progenitors via a transmembrane receptor. Erythropoietin is administered therapeutically to treat anemias
arising from chronic kidney failure, disorders of hematopoietic stem cells (myelodysplasia), or from the
collateral effects of chemical and radiologic treatments for cancer.
BIOMEDICAL IMPORTANCE
White blood cells, or leukocytes, serve as key sentries and potent defenders against invading pathogens.
Neutrophils, the most abundant type of white blood cell, ingest and destroy invading bacteria and fungi by a
process known as phagocytosis, while eosinophils phagocytize larger parasites. Circulating monocytes
migrate from the bloodstream to diseased tissues, where they differentiate into phagocytic macrophages.
Granulocytes such as basophils and mast cells release stored effectors that attract additional leukocytes to
the site of infection and trigger an inflammatory response. B lymphocytes generate and release protective
antibodies with the assistance of T lymphocytes. Other lymphocytes, such as cytotoxic T cells and natural
killer cells, target virally infected and malignantly transformed host cells. Malignant neoplasms of bloodforming tissues, called leukemias, can lead to the uncontrolled production of one or more of the major
classes of white blood cells. The hyperactivation of granulocytes during an allergic response can, in extreme
cases, lead to anaphylaxis and death. Leukopenia, a depression in the production of white blood cells, can
result from physical injury or infection of the bone marrow, chemotherapy, ionizing radiation, infection by
the Epstein-Barr virus (mononucleosis), an autoimmune response (lupus), or the displacement of bone
marrow cells by fibrous tissues (myelofibrosis). The resulting deficit in the levels of circulating leukocytes can
leave the affected individual vulnerable to infection (immunocompromised).
Leukocytes Migrate in Response to Chemical Signals
Leukocytes can be found throughout the body, migrating from the blood to sites of injury or infection in
response to chemical signals, a process referred to as chemotaxis. Migration out of the circulation takes
place via diapedesis, an amoeboid mechanism involving the cytoskeleton-mediated contortion of the cell. A
thin pseudopod is extended between the cells of the capillary epithelium. Once anchored on the other side,
cytoskeletal proteins squeeze the contents of the cell through the projection, which fills the distal end of the
pseudopod to form a new, translocated cell body, leaving behind the deflated remains. Once within the
tissues, locomotion proceeds via a similar, stepwise amoeboid mechanism.
Phagocytes Ingest Target Cells
White blood cells typically destroy invading microorganisms via phagocytosis. Phagocytic leukocytes
recognize and bind target cells using receptors that recognize bacterial lipopolysaccharides or
peptidoglycans. In most cases, however, infective pathogens are recognized indirectly, by the presence of
antibodies or complement factors that have previously adhered to their surface. The process of tagging an
invader with protective proteins to facilitate recognition by phagocytic leukocytes is called opsonization.
(A) The neutrophil binds an antigen molecule on the opsonized microbe via a receptor.
(B) The neutrophil envelops the microbe.
(C) Secretory granules fuse with the newly internalized phagosome, delivering their contents.
(D) Granule-derived enzymes and cytotoxins destroy the microorganism.
(E) The phagosome then fuses with the cell membrane, expelling any remaining debris.
Enzymes and Proteins of the Granules of Phagocytic Leukocytes
Phagocytic Leukocytes Generate Reactive Oxygen Species During the Respiratory Burst
Phagocytes employ ROS such as , and HOCl (hypochlorous acid) as a major component of the chemical and
enzymatic arsenal used to destroy ingested cells. Production of the various ROS takes place shortly (15-60
seconds) after internalization of an encapsulated cell, using O2 and electrons derived from NADPH. The
accompanying surge in oxygen consumption has been termed the respiratory burst.
LEUKOCYTES COMMUNICATE USING SECRETED EFFECTORS
The development of the immune and inflammatory responses by injured or infected tissues requires the
coordinated action of leukocytes and other cells. Much of this coordination is accomplished by secreting a
diverse set of small (< 25 kDa) proteins, termed cytokines, that includes interleukins, interferons, and
chemokines. The more than three dozen known interleukins derive their name from the cells in which they
are synthesized and from which they are secreted. They are generally designated by the class abbreviation IL
followed by an identifying number, eg, IL1, IL3, IL22. The interferons (IFN), on the other hand, derive their
name from their ability to inhibit, or interfere, with the replication of infecting viruses. Approximately 10
distinct families of interferons have been identified in animals to date. Chemokines attract and activate
migrating leukocytes to a site of injury or infection. Most cytokines are glycosylated. In general, they
stimulate both the leukocytes from which they are secreted (autocrine signaling) as well as other types of
leukocytes (paracrine signaling). Historically, cytokines have been distinguished from hormones by their
close association with immunity and inflammation.