Haemoglobin

Biomedical value

Heme-containing squirrels participate in processes of linkage and oxygen transport, in transport of electrons in photosynthesis. Detailed studying of haemoglobin taps a number of structural aspects, the general for many proteins. Speaking about the big biomedical value of these proteins, we mean, that the results received at research, visually illustrate structurally functional interrelations. Besides, these researches tap a molecular basis of some genetical diseases, such as a drepanocytic anaemia (resulting change of properties of a surface of b-subunit of haemoglobin) or a thalassemia (the chronic hereditable hemolitic disease characterised by disturbances of processes of synthesis of haemoglobin). The lethal effect of Cyanidum and carbonic oxide speaks that these materials quench physiological function of haemoproteins - cytochrome oxydase and haemoglobin accordingly. At last, stabilisation of quaternary structure of a deoxyhemoglobin 2,3-bifosfoglitseratom takes the central place in research of mechanisms of oxygen insufficiency in the conditions of high mountains and processes of acclimatisation to these conditions.

Chemical constitution

Chemically haemoglobin concerns bunch chrome of proteids. Its prosthetic bunch represents ferrum bond of protoporphyrin IХ, with molecular structure C34H32O4N4Fe and wears the name a heme. It gives to bond a coloration. The albuminous component of haemoglobin is called as a globin. The haemoglobin molecula contains 4 hemes and 1 globin. Amino acids are located in a globin in the form of four polypeptide chains; two of them are identical on structure, and them designate as an α-chain; two others too are identical among themselves and them designate as a β-chain. Hence, it is possible to express the globin formula as α-α/β-β or α2β2. α-polypeptide the chain consists from 141, β-polypeptide chain - of 146 amino acids.

Amino-acid structure and sequence (sequence) α - and β-chains. α - polypeptide the chain comes to an end with a combination of amino acids of valine - Leucinum, and β-polipeptidnaja a chain - a valine combination - Histidinum - Leucinum. α - and β - polypeptide chains in a haemoglobin molecula are not located linearly. Because of existence intramolecular forces, polypeptide chains are braided in the form of typical for proteins α - a helix spiral ("secondary structure"). Itself α - a helix spiral on everyone α - and β-polypeptide the chain is bent around regionally, forming plexuses of the ovoid form ("tertiary structure"). Separate parts α - helix spirals of polypeptide chains note letters from A to H.

All four is tertiary bent α - and β-polypeptide chains settle down regionally in a certain interrelation. They are bound among themselves not by the present chemical bonds, and intermolecular forces.

Four hemes of a haemoglobin molecula are located in the form of disks to honey by cords of four α - accordingly β-polypeptide chains, and each heme is bound to one polypeptide chain by means of coordination communication between Fe ++ - atom of a heme and histidinum the residual polypeptide chains.

The complex compounded of one heme and one α - β-polypeptide chains, is called unit svedberg. Obviously haemoglobin molecula consists of four unit svedberg. Now it is considered to be, that the molecular weight of haemoglobin is peer 64458, i.e. on one atom of iron, accordingly approximately on unit svedberg it is necessary on 16115.

Except the coordination communication existing between polypeptide chains of a globin, Fe ++ the atom of a heme has three more coordination communications. Two of them are bound by two nitrogenous atoms of the porphyrinic ring, and the third, in medium with low partial pressure of oxygen (venous blood), is bound to one molecula of water (the reduced haemoglobin). In medium with high partial pressure of oxygen (arterial blood), the third coordination communication is bridged to one molecula of oxygen, and bond - an oxyhemoglobin turns out. By continuous metamorphosis of an oxyhemoglobin into the reduced haemoglobin and anatropicly, transmission of oxygen from lungs to tissues is carried out.

Kinetics of hemoglobin oxygenation

Haemoglobin binds four moleculas of oxygen on tetramer (on one on a heme in each subunit); especially important it is distinguished it from a myoglobin is the curve of saturation oxygen which has the sigmoidal form. Thus, ability of haemoglobin to bind oxygen depends on, whether other moleculas of oxygen contain in the given tetramer. If yes, the subsequent moleculas of oxygen join easier. Hence, for haemoglobin the kinetics of co-operative linkage thanks to which it binds an oxygen maximum quantity in lungs is characteristic and gives an oxygen maximum quantity at those partial pressures of oxygen which take place in peripheric tissues.

Affinity of hemoglobins to oxygen is characterised by size Р50 - value of partial pressure of oxygen at which semisaturation of haemoglobin by oxygen is observed. Value Р50 at at different organisms is essential differs, but in all cases it exceeds value of partial pressure of oxygen in peripheric tissues of a surveyed organism. It well illustrates foetal haemoglobin of the human (НВF). For HbA Р50=26 mm Hg, and for HbF Р50=20 mm Hg Thanks to this difference haemoglobin F culls oxygen at HbA, being in placental blood. However after a birth of child HbF loses the function; possessing higher affinity to oxygen, it liberates its smaller quantity in tissues.

The oxygenation ia accompanied BY appreciable conformational changes in hemoglobin.

Oxygen linkage is accompanied by a breakage of the saline communications formed by trailer carboxylic bunches of subunits. It facilitates linkage of following moleculas of oxygen as the breakage of smaller number of saline communications thus is required. The specified changes considerably influence secondary, tertiary and especially quaternary structure of haemoglobin. Thus one α/β - pair subunits turns concerning another α/β - pairs that leads to reduction of tetramer and rising of affinity of hemes to oxygen.

Conformational changes in haemobunch environment

The haemoglobin oxygenation is accompanied by structural changes in a haemobunch environment. At an oxygenation the atom of iron which in a deoxyhemoglobin acted on 0,06 nanometers from a plane heme rings, involves in this plane. After atom of iron is closer to a heme proximal Histidinum (F8), and also the next residual bound to it moves.

Transport of carboneum dioxide

Haemoglobin not only tolerates oxygen from lungs to peripheric tissues, but also accelerates transport of a carbon dioxide from tissues to lungs. Haemoglobin binds a carbon dioxide right after oxygen liberation; approximately 15 % of the carbon dioxide which is present at blood, are tolerated by haemoglobin moleculas. The carbonic anhydrase being in erythrocytes catalyzes metamorphosis of a carbon dioxide arriving from tissues into coal acid. Coal acid quickly dissociates on bicarbonate-ion and a proton, and balance is moved towards dissociation. For prevention of dangerous rising of acidity of blood there should be a buffer system, capable to absorb excess of protons. Haemoglobin binds two protons on each four released moleculas of oxygen and defines buffer capacity of blood. In lungs there is an anatropic process: Apposition of oxygen to a deoxyhemoglobin is accompanied by liberation of protons which contact bicarbonates-ions, translating them in coal acid. Further effectively reacting carbonic anhydrase catalyzes metamorphosis of coal acid into a carbon dioxide exhaled from lungs. Thus, oxygen linkage is closely interfaced to a carbon dioxide expiration. This reversible phenomenon is known as Bohr effect. Bohr effect is property tetra shaped haemoglobin and the heme - hemed is defined by the interaction underlying co-operative effects.

Molecular basis of Bohr effect

The protons responsible for Bohr effect, are liberated as a result of destruction of saline ponticuluses by which linkage of oxygen with T - structure is accompanied; they are disconnected from atoms of nitrogen of the residual of Histidinum (146) in β - chains. These protons shift balance towards formation of coal acid which is disjoined by a carbonic anhydrase with carbon dioxide formation.

On the contrary, at oxygen liberation again it is formed T - structure with saline ponticuluses inherent in it at which formation there is an apposition of protons to Histidinum residual in β - chains. Thus, in peripheric tissues protons favour to formation of saline ponticuluses by a protonation (on atom of nitrogen) the trailer residual of Histidinum in β - subunits. Formation of saline ponticuluses forces remission of oxygen from oxyogenised R - haemoglobin forms. So, rising of concentration of protons promotes oxygen remission, and rising of concentration of oxygen suscitates liberation of protons. The first of these effects shows in oxygen-dissociation curve alteration to the right at rising of concentration of ions of hydrogen (protons).

Hemoglobin concentration

Normal concentration of haemoglobin at the adult human from 80 to 115 % (conditional protsentov=13,0-18,5). For average size accept 100 % (=16). Normal amounts at men approximately on 10 % above (90-115 %, according to 14,5-18,5 of haemoglobin), than at women (80-100 %, according to 13-16 of haemoglobin).

Normal concentration of haemoglobin at the child is essential differs from norms at the adult. These features are shown on tab. 1.

Table 1

Average concentration of haemoglobin in blood in the seasons of children's age. The maximum fluctuations of average sizes +/-1-2 %

Haemoglobin in a blood plasma

Normal plasma contains the traces of haemoglobin which are not exceeding 10 mg. At an intravital hemolysis concentration of haemoglobin in plasma raises. Moderate risings (to 25mg.) meet at immune hemolitic anemias, a Cooley anaemia, hemoglobinosis C, a drepanocytosis, etc. Strong augmentations (over 100 mg.) meet at all hemoglobinurias.

Research means

Many methods of definition of concentration of haemoglobin were offered. The major bunches of methods the following:

1. Colorimetric methods. Haemoglobin colorimetre as the oxyhemoglobin or the reduced haemoglobin or at first transform it into colour derivatives (a hydrochloride hematin, alkaline haemoglobin, a methemoglobin, a carboxyhemoglobin, cyanogen haemoglobin, azide - a methemoglobin and so forth). Here it is possible to carry and the first method for the haemoglobin definition, offered Welcker in 1854 at which colour of a drop of blood on a filter paper compare to a series of colour paper standards. On the basis of metamorphosis of haemoglobin into a hydrochloride hematin and the changes bound to it in electrical conductivity, Kneller has offered an electronic method of definition of concentration of haemoglobin.
2. Gazometrichesky methods. Haemoglobin sate with gas, for example oxygen, carbonic oxide (CO). By quantity of absorbed gas judge quantity of haemoglobin. Quantity of oxygen establish Van Slyke Instrument, Barcroft apparatus or any other apparatus for oxygen definition.
3. The methods based on definition of iron in a haemoglobin molecula. As the haemoglobin molecula contains precisely certain quantity of iron (0,0347 %), by its quantity the quantity of haemoglobin is established also.

Methemoglobin

Methemoglobin - derivative haemoglobin in which the divalent atom of iron passes in the trivalent. At exchange processes in erythrocytes known quantities of a methemoglobin which, however, is recovered anatropicly in haemoglobin under the influence of methemoglobin reductase enzyme so in integral blood of the healthy human the methemoglobin does not exceed 2 % of the general haemoglobin content (0,03-0,3) are always formed.

Sulfhemoglobin

The chemical structure sulfhemoglobin is not found out. Possibly, two vinyl bunches of haemoglobin are bridged, by means of SО2-мостиков, with the next methine communications. In norm, sulfhemoglobin in blood is not present. It appears at venenatings with antimony bonds, Phenacetinum, bromine, sulfonamides, Sodium nitritums (well water), sulfuric bonds and so forth

Definition sulfhemoglobin in blood it is possible to effect spectroscopicallies. The sulfhemoglobin spectrum does not variate from gaining of sulphide of ammonium, but disappears from gaining Na2S2О4 and 2 ml of 10 % of caustic soda, or several drops of peroxide of hydrogen of 3 %.

Hemoglobin types

Recently still was considered, that haemoglobin of the adult human represents the unique bond. That in embryonal life there is the especial type of haemoglobin named HbF, in 155 times steadier to n/12 was known only to sodium alkali, than normal haemoglobin. Recently, thanks to works Pauling and its employees, etc., it was found out, that haemoglobin of the adult human both at normal, and at morbid conditions does not represent a homogeneous chemical compound. Normal and pathological types of haemoglobin which have presented an exchange of haemoglobin in new light have been open many and have specified pathes for research of a pathogenesis of some anemias. It has been established, that at some diseases the special types of haemoglobin, characteristic for the given anaemia are observed. Haemoglobin types are of great importance not only for the diagnosis, but also alternate a question on a pathogenesis of an anaemia from purely morphological range in the biochemical. The anemias caused by appearance of a pathological type of haemoglobin, are called as hemoglobinopathies or hemoglobinoses.

It was found out, that the human has three basic types of normal haemoglobin: embryonal U, foetal - F and haemoglobin of the adult human - A. HbU (it is named under the initial letter of a word uterus) meets in an embryos between 7 and 12 weeks of life, then it disappears and there is a foetal haemoglobin which after the third month is the basic haemoglobin of a foetus. After this there is gradually ordinary haemoglobin of the adult human named Hb A. The quantity of foetal haemoglobin gradually decreases, so at the moment of a birth of 80 % of haemoglobin represents Hb A and only 20 % - HbF. After a birth foetal haemoglobin continues to decrease and by 2-3 year of life compounds only 1-2 %. Too quantity of foetal haemoglobin and at the adult. Quantity HbF exceeding 2 % is considered pathological for the adult human and for children is more senior 3 years.

Except normal types of haemoglobin now it is known over 50 its pathological variants. They at first have been named by Latin letters. The letter In in notations of types of haemoglobin is absent, since by it is designated pristinely Hb S. It was soon found out, that letters of the αbet will not suffice for notation of all pathological types of haemoglobin. Therefore began to apply for this purpose names of patients, hospitals, laboratories, names of places and districts. The most convenient is the nomenclature on a constitutional formula (see more low).

Both normal, and pathological types of haemoglobin differ not on structure of the protoporphyrinic ring, and globin construction. The difference can consist in change of the whole pairs polypeptide chains in a haemoglobin molecula, or at conservation of the same polypeptide chains, are substituted on a certain place in primary structure one amino acid another.

The first possibility meets at hemoglobins H, F, Barths, А2 and U. Instead of normal structure of haemoglobin A - α-α/β-β, accordingly α2/β2, haemoglobin H has structure β-β-β-β, accordingly β4, that means, that on both α-polypeptide chains are substituted by new two β-polypeptide chains. Hemoglobins F, Barths and А2 have two new chains, designated scale and delta, and at haemoglobin U new a chain, designated Ipsilonum. Structure HbF α-α/γ scale, accordingly α2/γ2, structure of haemoglobin Barths scale-scale-γ scale, corresponds γ4, structure HbA2 an α-α/delta-delta, accordingly α2/γ2, structure of haemoglobin U - α-α/IPSILONUM-Ipsilonum, accordingly alfa2/ipsilon2.

Pathological hemoglobins which consist of four identical polypeptide chains, designate tetramers. Tetramers α4 and δ4 in vivo were not observed till now.

The second possibility meets at the majority of types of haemoglobin. So for example the unique difference between HbS and HbA consists that on 6th place in β-polypeptide chains instead of a glutamine there is a valine, a unique difference between HbI and HbA that on 16th place in α-polypeptide chains the lysine is substituted by asparaginic acid.

When anomaly consists in amino acid replacement in α-polypeptide chains speak about α-anomaly when consists in β-polypeptide chains - about β-chain anomaly when in γ polypeptide chains - about γ chain anomaly (pathological variants HbF) and when in delta-chain - about anomaly delta-chain (pathological variants HbA2).

At studying of haemoglobin types the question on structure of globins is of great importance. On the one hand, the structure is the most true mean of differentiation of separate types of haemoglobin one from another, possibility for drawing up of strictly scientific nomenclature of the last on the other hand is framed.

Methods of hemoglobin kinds differentiation

1. For differentiation of separate types of human haemoglobin use an electrophoresis on starch block, on amylaceous gel, on agar gel, on cellulose acetate sheets, on acrylamide gel, on carboxymethyl cellulose gel, an electrophoresis at a current high voltage.
2. The method second for value which use now for differentiation of separate kinds of haemoglobin, the chromatography is. Especially good results it turns out at the use as an ion-exchange resin adsorbent amberlith and ion-exchange dextrans gel.
3. For differentiation of some kinds of haemoglobin use also their solubility in some dissolvents. The most known test of this bunch is assay Itano for the proof of presence HbS. At this assay we are served by that circumstance, that reduced HbS precipitates in 2,24 m the buffer, contrary to other types of haemoglobin. This assay matters in particular for differentiation HbS and HbD because, HbS and HbD possess identical electrophoretic and chromatographic motility.
4. For difference HbA from HbF use, as it has been underlined above, fastness at a denaturation by solutions of sodium alkali.
5. Bunch F hemoglobins (HbF, Hb Fellas, Hb Alexander and Hb Barths) differs from other haemoglobin types and on the characteristic tryptophan strip at 289,8 nanometers of an ultra-violet spectrum. The hemoglobins possessing bunch of M, have no absorption strip at a wavelength of 630 nanometers, but show the enlarged absorption at 600 nanometers.
6. "Fingerprint method". Business concerns the major method of an establishment of "primary structure" haemoglobin at various haemoglobin types. Investigated haemoglobin hydrolyze Trypsinum at what polypeptide chains globin moleculas break up to the big number of peptides. The peptide admixture is subjected by electrochromatographies on a paper, i.e. in one direction chromatographic separation is spent electrophoretic, in other. Turn out characteristic for separate types of hemoglobins of an electrochromatogram on which they can be distinguished precisely. Definition of amino-acid structure of separate peptides gives the chance primary structure of a globin of the conforming haemoglobin type. Doing analogy with corresponding on complexity and accuracy criminalistic technics for studying of impresses of dactyls of arms, it has been named "fingerprint" method.
7. For definition of structure of polypeptide chains in any haemoglobin type it is possible to take advantage and so-called "recombination" or a "hybridization" method. If to admix known and unknown haemoglobin at pH 4,3, they dissociate the semimoleculas consisting of the conforming pairs of polypeptide chains. After solution neutralisation polypeptide pairs are again combined in the whole haemoglobin moleculas at what can it will turn out also new "hybrid" haemoglobin moleculas. Their identification by an electrophoretic mean or a chromatography will allow to make the conclusion about polypeptide structure of an unknown haemoglobin type. This method is designed also mainly for the scientific research purposes.
8. Immunologic methods
9. Except the above-stated methods at differentiation of separate types of haemoglobin use also differences in a crystalline constitution, an isoelectric point etc.
10. Methods of the cytologic definition of a type of haemoglobin in erythrocytes on a blood smear are developed also. So in erythrocytes it is possible to prove presence HbF by processing of a bloody smear citric acid a buffer admixture with pH 3,2-3,6. Under these conditions HbA it is taken also erythrocytes in which it prevailed, remain only in a kind eritrocite shades whereas HbF remains also the erythrocytes containing mainly this type of haemoglobin, keep the maintenance.

Hemoglobin at the drepanocytic anemia

In haemoglobin S residual Glu А2 (6) β it is substituted on Val. Residual А2 (Glu or Val) settles down on a surface of a molecula of haemoglobin and contacts in water, and replacement polar residual Glu on not polar Val leads to appearance on a surface β-subedenitsy "a sticky field". This sticky field is present both in oxyogenised, and at deoxygenated haemoglobin S (in haemoglobin A is absent). On a surface of deoxygenated haemoglobin there is a complementary field, capable strongly to contact a sticky field of β-subunit whereas in the oxyogenised haemoglobin this field masks other bunches. When haemoglobin S passes in a deoxygenated condition, its sticky field contacts a complementary field on other molecula of deoxygenated haemoglobin. There is a polymerisation of deoxyhemoglobin S and its sedimentation in the form of long fibers. Fibers of deoxyhemoglobin S mechanically deform an erythrocyte, betraying it the crescent form that leads to a killing and set of secondary clinical implications. Thus, if it was possible it is possible to sustain haemoglobin S in the oxyogenised condition or at least to reduce to a minimum concentration of deoxygenated haemoglobin S we would manage to prevent polymerisation of deoxygenated haemoglobin S and formation of "crescent" cells. Clearly, that the T-form of haemoglobin S is subject to polymerisation. It is interesting to notice (though in the practical plan it unimportantly), that the ferri-ion of methemoglobin A remains in a plane of the porphyrinic ring and by that stabilises the haemoglobin R-form. The same concerns and haemoglobin at a drepanocytic anaemia: haemoglobin S in a ferri-condition (methemoglobin S) is not subject to polymerisation as it is stabilised in the R-form.

In deoxyhemoglobin A too there is a receptor field, capable to co-operate with a sticky field of oxyogenised or deoxygenated haemoglobin S, but to to deoxyhemoglobin A it is not enough apposition of "sticky" haemoglobin S for polymer formation as deoxyhemoglobin A of a sticky field does not contain and cannot bind the following molecula of haemoglobin. Hence, linkage of deoxyhemoglobin A with R - or the T-form of haemoglobin S blocks polymerisation.

As a result of polymerisation of deoxyhemoglobin S spiral fibrillar structures are formed. Thus each molecula of haemoglobin contacts to four next moleculas. Formation of similar tubular fibers responsibly for mechanical disturbances in an erythrocyte containing them: it gets the crescent form, becomes subject to a lysis at the moment of transit of clefts by it in lien sinusoids.

Thalassemias

Other important bunch of the disturbances bound to anomalies of haemoglobin - thalassemias. For them the lowered rate of synthesis of α-chains of haemoglobin (α-talassemija) or β-chains (β-talassemija) is characteristic. It leads to an anaemia which can accept very serious form. Last years will reach notable progress in finding-out of the molecular mechanisms responsible for development of a thalassemia.