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

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.

Blood Groups

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.

Iron's oxidation state in oxyhemoglobin

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 O₂^2-. 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 of ligands

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 (S^2-), 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.^[]

Degradation of hemoglobin in vertebrate animals

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

Role in disease

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).

Diagnostic use

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 A_1c 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 A_1c runs 6.0% or less show good longer-term glucose control. Hb A_1c 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 (HCO^3-):

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.

Buffering hides from view the real change in H⁺ that occurs.

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 Body Buffer Systems
Site	Buffer System	Comment
ISF	Bicarbonate	For metabolic acids
	Phosphate	Not important because concentration too low
	Protein	Not important because concentration too low
Blood	Bicarbonate	Important for metabolic acids
	Haemoglobin	Important for carbon dioxide
	Plasma protein	Minor buffer
	Phosphate	Concentration too low
ICF	Proteins	Important buffer
	Phosphates	Important buffer
Urine	Phosphate	Responsible for most of 'Titratable Acidity'
	Ammonia	Important - formation of NH4^+
Bone	Ca carbonate	In prolonged metabolic acidosis

The Bicarbonate Buffer System

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]

The concentration of H₂O is so large (55.5M) compared to the other components, the small loss of water due to this reaction changes its concentration by only an extremely small amount. This means that [H₂O] is effectively constant. This allows further simplification as the two constants (Ka and [H₂O] ) can be combined into a new constant K’a.

K’a = Ka x [H₂O] = [H⁺] . [HCO₃-] / [CO₂]

Substituting:

K'a = 800 nmol/l (value for plasma at 37C)

[CO₂] = 0.03 x pCO₂ (by Henry’s Law) [where 0.03 is the solubility coefficient]

into the equation yields the Henderson Equation:

[H⁺] = (800 x 0.03) x pCO₂ / [HCO₃-] = 24 x pCO₂ / [HCO₃-] nmol/l

Taking the logs (to base 10) of both sides yields the Henderson-Hasselbalch equation:

pH = log10(800) - log (0.03 pCO₂ / [HCO₃-] )

pH = 6.1 + log ( [HCO₃] / 0.03 pCO₂ )

On chemical grounds, a substance with a pKa of 6.1 should not be a good buffer at a pH of 7.4 if it were a simple buffer. The system is more complex as it is ‘open at both ends’ (meaning both [HCO₃] and pCO₂ can be adjusted) and this greatly increases the buffering effectiveness of this system. The excretion of CO₂ via the lungs is particularly important because of the rapidity of the response. The adjustment of pCO2 by change in alveolar ventilation has been referred to as physiological buffering.

The bicarbonate buffer system is an effective buffer system despite having a low pKa because the body also controls pCO₂

Other Buffers

The other buffer systems in the blood are the protein and phosphate buffer systems.

These are the only blood buffer systems capable of buffering respiratory acid-base disturbances as the bicarbonate system is ineffective in buffering changes in H⁺ produced by itself.

The phosphate buffer system is NOT an important blood buffer as its concentration is too low

The concentration of phosphate in the blood is so low that it is quantitatively unimportant. Phosphates are important buffers intracellularly and in urine where their concentration is higher.

Phosphoric acid is triprotic weak acid and has a pKa value for each of the three dissociations:

pK_a1 = 2

pK_a2 = 6.8

pK_a3 = 12

H₃PO₄

<= = = >

H⁺ + H₂PO₄^-

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

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.

Blood Groups

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.

Iron's oxidation state in oxyhemoglobin

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 O₂^2-. 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 of ligands

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 (S^2-), 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.^[]

Degradation of hemoglobin in vertebrate animals

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

Role in disease

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).

Diagnostic use

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 A_1c 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 A_1c runs 6.0% or less show good longer-term glucose control. Hb A_1c 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 (HCO^3-):

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.

Buffering hides from view the real change in H⁺ that occurs.

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 Body Buffer Systems
Site	Buffer System