HEMOGLOBIN, Hb (haemoglobinum; Greek haima blood + lat. globus a ball) — a hemoprotein, the complex protein relating to gemsoderzhashchy chromoproteids; carries out transfer of oxygen from lungs to fabrics and participates in transfer of carbon dioxide gas from fabrics in a respiratory organs. Contains in erythrocytes of all vertebrata and some backboneless animals (worms, mollusks, arthropods, erinaceouses), and also in root nodules of some bean plants. Pier. the weight (weight) of G. of erythrocytes of the person is equal to 64 458; is in one erythrocyte apprx. 400 million Molecules. In water G. we will well dissolve, it is insoluble in alcohol, chloroform, ether, well crystallizes (the form of crystals of G. of various animals is not identical).
Simple protein — a globin and ferriferous prosthetic (nonprotein) group — gems is G.'s part (96 and 4% of the mass of a molecule respectively). At pH lower than 2,0 there is a splitting of a molecule G. on gems and a globin.
Gems (C 34 H 32 O 4 N 4 ) represents zhelezoprotoporfirin — complex compound of protoporphyrin IX with bivalent iron. Iron is in the center of a protoporphyrinic kernel and is connected with four nitrogen atoms of pyrrol kernels (fig. 1): two communications coordination and two communications with substitution of hydrogen.
As the coordination number of iron is equal 6, two valencies remain unused, one of them is implemented during the binding gem with a globin, and oxygen or other ligands — CO, F joins the second + , azides, water (fig. 2) etc.
A complex of protoporphin IX with Fe 3+ call hematin. Muriatic salt of hematin (chlorhemin, hemin) is easily emitted century a crystal look (so-called crystals of Teykhmann). Gem has ability to form complex connections with nitrogenous compounds (ammonia, pyridine, a hydrazine, amines, amino acids, proteins etc.), turning at the same time in hemochromogens (see). As at all animal species of gems it is identical, distinctions in properties of haemo globins are caused by features of a structure of a proteinaceous part of a molecule G. — a globin.
Globin — protein like albumine, contains four polypeptide chains in the molecule: two alpha chains (about 141 amino-acid rests enter each of which) and two beta chains containing 146 remains of amino acids. Thus, the proteinaceous component of a molecule G. is constructed of 574 remains of various amino acids. Primary structure, i.e. genetically caused sequence of an arrangement of amino acids in polypeptide chains of a globin of the person and a number of animals, is completely studied. Distinctiveness of a globin of the person is absence in its composition of amino acids from a leucine and cystine. The N-trailer remains in alpha and beta chains are the remains of valine. The S-trailer remains of alpha chains are presented by the remains of arginine, and beta chains — a histidine. Penultimate position in each of chains is held by the remains of tyrosine.
The X-ray crystallographic analysis of crystals of G. allowed to reveal the main features of spatial structure of its molecule [Perutts (M. of Perutz)]. It turned out that alpha and beta chains contain spiral segments of various length which are constructed by the principle of alpha spirals (secondary structure); the alpha chain has 7, and a beta chain — 8 spiral segments connected by not spiral sites. Spiral segments, since the N-end, are designated by letters of the Latin alphabet (And, B, C, D, E, F, G, N), and not spiral sites or tilt angles of spirals have the corresponding designation (AB, BC, CD, DE etc.). Not spiral sites on amine (N) or carboxyl (C) the end of a chain of a globin designate according to NA or NANOSECOND. The amino-acid remains are numbered in each segment and, besides, in brackets numbering of this rest from the N-end of a chain is given.
Spiral and not spiral sites are definitely laid in space that defines tertiary structure of chains of a globin. The last is almost identical at alpha and G.'s beta chains, despite significant differences in their primary structure. It is caused by the specific arrangement of polar and hydrophobic groups of amino acids leading to accumulation of unpolar groups in the interior of a globule with formation of a hydrophobic kernel. Polar groups of protein are turned to an aqueous medium, being with it in contact. In each chain of a globin near a surface there is a hydrophobic hollow («a gemovy pocket»), in a cut is located gems, being guided so that his unpolar deputies are sent to inside molecules, being a part of a hydrophobic kernel. Results apprx. 60 unpolar contacts between gemy and a globin and one-two polar (ionic) contacts gem with alpha and beta chains in which the remains propionic participate to - you are gem, effluent of hydrophobic «pocket». An arrangement gem in a hydrophobic hollow of a globin provides a possibility of reversible oxygenation to Fe 2+ gem without oxidation of the last to Fe 3+ also it is characteristic of haemo globins of different types of animals. Confirmation of it is extreme sensitivity of G. to any changes of unpolar contacts close gem. So, replacement gem in G. on haematoporphyrin leads to sharp disturbance of properties.
Some amino-acid remains surrounding gems in a hydrophobic hollow are among invariant amino acids, i.e. the amino acids identical to different types animal and essential to Function. Among invariant amino acids the great value is allocated for three: to the remains of a histidine, so-called proximal histidines (the 87th position in and - and the 92nd position in P-chains), to distal histidines (the 58th position in and - and the 63rd position in (5 chains), an also to the rest of E-11 valine (the 62nd position in an alpha chain and the 67th position in a beta chain).
Communication between a so-called proximal histidine and iron gem is the only chemical communication between them (the fifth coordinate bond of Fe atom is implemented 2+ gem) and directly influences oxygenation to gem. The «distal» histidine is directly not connected with gemy and does not take part in fixation of oxygen. Its value consists in stabilization of Fe atom 2+ against irreversible oxidation (apparently, due to hydrogen bonding between oxygen and nitrogen). The rest of valine (E-11) is some kind of speed governor of oxygenation to gems: in beta chains it is sterically located so that takes that place where oxygen shall join owing to what oxygenation begins with flfa-chains.
A proteinaceous part and prosthetic group of a molecule G. exert at each other strong impact. A globin gem changes many properties, giving it ability to binding of oxygen. Gem provides resistance of a globin to action to - to t, heating, splitting with enzymes and causes features of crystallizational Properties.
Polypeptide chains with the molecules attached to them gem form four main parts — subunits of a molecule. The nature of connection (laying) them among themselves c an arrangement in space define features of quarternary structure of G.: and - and P-chains are located on corners of a tetrahedron around an axis of symmetry, and alpha chains lie over p-chains and as if squeeze between them, and all four gem are far removed from each other (fig. 3). In general the tetrameric spheroid particle with sizes of 6,4 X 5,5 x 5,0 nanometers is formed. The quarternary structure is stabilized by salt bonds between α — α-and β-β-tsepyam and two types of contacts between α and β-chains (α1-β1 and α2-β2). Contacts α1-β1 are most extensive, 34 amino-acid rests, the majority of interactions not polarly participate in them. The contact α1-β2 includes 19 amino-acid remains, the majority of bonds also not polarly, except for several hydrogen bindings. All remains which are in this contact are identical at all studied animal species while 1/3 remains in α1-β1-контактах vary.
Of the person geterogenen that is caused by distinction of the polypeptide chains which are its part. So, G. of the adult making 95 — 98% of G. of blood (HbA) supports two α-and two β-chains; the small fraction G. (HbA2) reaching the maximum content of 2,0 — 2,5% supports two α-and two σ-chains; hemoglobin of a fruit (HbF), or the fetalis hemoglobin making 0,1 — 2% in blood of the adult consists of two α-and two γ-chains.
Fetalis G. is replaced with HbA in the first months after the birth. It is characterized by considerable resistance to a thermal denaturation what methods of determination of its content in blood are based on.
Depending on structure of polypeptide chains the listed types G. are designated as follows: HbA — as Hbα2β2, HbA2 — as Hbα2σ2, a HbF — as Hbα2γ. At congenital anomalies and diseases of the hemopoietic device abnormal types G., napr appear, at drepanocytic anemia (see), thalassemias (see), an inborn methemoglobinemia of not euzymatic origin (see. Methemoglobinemia ), etc. Most often substitution of the only amino acid in one couple of polypeptide chains meets.
Depending on the size of valency of atom of iron gem and like a ligand in a molecule G. the last can be in several forms. The recovered G. (dezoksi-Hb) has Fe 2+ with a free sixth valency, at accession of O to it 2 the oxygenic form G. is formed (HbO 2 ). At action on HbO 2 a number of oxidizers (ferricyanide of potassium, nitrites, quinones, etc.) there is an oxidation of Fe 2+ to Fe 3+ with education methemoglobin, O, incapable of transfer 2 . Depending on the size pH of the environment distinguish an acid and alkaline form of a methemoglobin, H containing in quality of the sixth ligand 2 O or OH group. In blood of healthy people concentration of a methemoglobin makes 0,83+0,42%.
The methemoglobin has ability to strongly connect hydrogen fluoride, hydrocianic to - that and other substances. Use this its property in medical practice for rescue of the people poisoned hydrocianic to - that. Various derivative G. differ on absorption spectrums (tab).
Functional properties of hemoglobin. The main biol, G.'s role — participation in gas exchange between an organism and external environment. Provides transfer with blood of oxygen from lungs to fabrics and transport of carbonic acid from fabrics to lungs (see. Gas exchange ). Also buffer properties G., forming the powerful gemoglobinny and oksigemoglobinny buffer systems of blood promoting, thus, maintenance of acid-base equilibrium in an organism are not less important (see. Buffer systems , Acid-base equilibrium ).
Oxygen capacity of HbO 2 makes 1,39 ml of O 2 on 1 g of HbO 2 . G.'s ability to connect and give oxygen is reflected its oxygen dissotsiatsionnoy curve (ODC), characterizing percent of saturation G. oxygen depending on the partial pressure of O 2 (pO 2 ).
Tetrameric molecules G. have the S-shaped form of CDC that demonstrates that G. provides optimum binding of oxygen with its rather low partial pressure in lungs and return — with rather high partial pressure of oxygen in fabrics (fig. 4). The maximum return of oxygen to fabrics is combined with preservation of its high partial pressure in blood that provides penetration of oxygen into depth of fabrics. The size of partial pressure of oxygen in mm of mercury., at a cut of 50% of G. it is oxygenated, is a measure of affinity of G. to oxygen and P50 is designated.
Oxygenation to four gems G. happens consistently. The S-shaped nature of CDC of G. demonstrates that the first molecule of oxygen connects to G. very slowly, i.e. its affinity to G. is small as it is required to break off salt contacts in a molecule of a dezoksigemoglobin. However accession of the first molecule of oxygen increases affinity to it the remained three gem, and further oxygenation of G. happens much quicker (oxygenation of the fourth gem occurs by 500 times quicker, than the first). Therefore, cooperative interaction between centers, connecting oxygen is available. Patterns of reaction of G. with carbon monoxide (SO) the same, as for oxygen, but G.'s affinity to WITH is nearly 300 times above, than to O 2 , what causes high virulence of carbon monoxide gas. So, at concentration WITH in air, equal 0,1%, more than a half of G. of blood are connected not with oxygen, and with carbon monoxide gas. At the same time there is a formation of the carboxyhaemoglobin incapable of transfer of oxygen.
Regulators of process of oxygenation of hemoglobin. The great influence on processes of oxygenation and deoxygenation is rendered by hydrogen ions, organic phosphates, inorganic salts, temperature, carbonic acid and some other substances controlling the size of affinity of G. to oxygen according to fiziol. requests of an organism. Dependence of affinity of G. to oxygen from the size pH of the environment carries the name of Bohr effect (see. Verigo effect ). Distinguish «acid» <(rn6) and «alkaline» Bohr effect >(pH6). The greatest fiziol. «alkaline» Bohr effect matters. Its molecular mechanism is caused by existence in a molecule G. of a number of positively charged functional groups which dissociation constants are much higher in the dezoksigemoglobena due to formation of salt bridges between negatively charged groups of the next proteinaceous chains in a molecule. In the course of oxygenation owing to the occurring conformational changes of a molecule G. salt bridges collapse, pH of negatively charged groups and protons changes are allocated in solution. Therefore, oxygenation leads to eliminating of a proton (H + ) from a molecule G. and, on the contrary, change of the size pH, i.e. indirectly ion concentrations of H + , Wednesdays influences accession to G. of oxygen. Thus, H + becomes the ligand communicating preferential with dezoksigemoglobiny and by that reducing its affinity to oxygen, i.e. change of the size pH in the acid party causes shift of CDC to the right. Process of oxygenation is endothermic, and temperature increase promotes eliminating of oxygen from a molecule. Therefore, strengthening of activity of bodies and temperature increase of blood will cause shift of CDC to the right, and return of oxygen to fabrics will increase.
A peculiar regulation of process of oxygenation is carried out by the organic phosphates which are localized in erythrocytes. In particular, 2,3 diphosphoglycerate (DFG) considerably reduce G.'s affinity to oxygen, promoting eliminating of O 2 from oxyhemoglobin. Influence of DFG on G. increases at reduction of a pH value (in limits fiziol, areas) therefore its influence on G.'s CDC is shown more at the low sizes pH. DFG communicates preferential with dezoksigemoglobiny in molar ratios 1:1, entering an internal hollow of its molecule and forming 4 salt bridges from two alpha NH 2 - groups of the remains of valine of beta chains and, apparently, with two imidazolny groups of histidines N-21 (143) beta chains. Influence of DFG decreases with temperature increase, i.e. process of linkng of DFG with a molecule G. is exothermic. It leads to the fact that in the presence of DFG dependence of process of oxygenation on temperature considerably disappears. Therefore, normal release of oxygen blood becomes possible in a wide interval of temperatures. Similar effect, though to a lesser extent, ATP, pyridoxal phosphate render other organic phosphates. Thus, concentration of organic phosphates in erythrocytes has considerable effect on respiratory function G., quickly adapting it for various fiziol, and patol, to the conditions connected with disturbance of oxygenation * (change of the oxygen content in the atmosphere, blood loss, regulation of transport of oxygen from mother to a fruit through a placenta, etc.). So, at anemia and a hypoxia in erythrocytes the maintenance of DFG increases that the CDC shifts to the right and causes big return of oxygen to fabrics. Many neutral salts (acetates, phosphates, potassium chlorides and sodium) also reduce G.'s affinity to oxygen. This effect depends by nature substances and is similar to effect of organic phosphates. In the presence of high concentration of salt G.'s affinity to oxygen reaches a minimum — to the same extent for various salts and DFG, i.e. both salts, and DFG compete with each other for the same centers of binding on a molecule. So, e.g., influence of DFG on G.'s affinity to oxygen disappears in the presence of 0,5 M of sodium chloride.
In 1904 Bor (Ch. Bohr) with sotr. showed reduction of affinity of G. to oxygen at increase in partial pressure of carbon dioxide gas in blood.
Increase in content of carbon dioxide gas leads first of all to change of pH of the environment, however P50 value decreases more than it should be expected at such reduction of a zn
of a cheniye of pH. It is caused by specific relationship of carbon dioxide gas with not loaded with alpha NH2 - groups of alpha chains, and it is possible, and G.'s beta chains with formation of carbamates (carbohemoglobin) according to the following scheme:
HbNH 3 + <-> HbNH 2 + H +
HbNH 2 + CO 2 <-> HbNHCOO - + N +
Dezoksigemoglobin connects bigger amount of carbon dioxide gas, than HbO 2 . In an erythrocyte presence of DFG competitively inhibits formation of carbamates. By means of the carbamate mechanism about 15% of carbon dioxide gas are removed from an organism of healthy people at rest. More than 70% of buffer capacity of blood are provided to G. which is in it that leads also to considerable indirect participation of G. in transfer of carbon dioxide gas. At course of blood through HbO fabrics 2 passes in dezoksigemoglobin, connecting at the same time ions of H+ and translating thereby H 2 CO 3 in HCO 3 - . Thus, with direct and indirect participation of G. more than 90% of the carbonic acid coming from fabrics to blood communicate and it is transferred to lungs.
It is essential that all specified regulators of shift of CDC (H + , DFG, CO 2 ) are interconnected among themselves that is of great importance at a row arising patol, states. So, increase in concentration of DFG in erythrocytes is result of complex changes in their metabolism, in Krom increase in a pH value is the main condition. At acidosis and an alkalosis also owing to interrelation between H + and DFG occurs alignment of size P 50 .
Metabolism of hemoglobin
G.'s Biosynthesis happens take part in young forms of erythrocytes (erythroblasts, normoblasts, reticulocytes) where the atoms of iron included in G. V structure synthesis of a porphyrinic ring get glycine and amber to - that with education δ-aminolevulinic to - you to - you to - you... Two molecules of the last turn into pyrrol derivative — the predecessor of porphyrine. The globin is formed of amino acids, i.e. in the regular way synthesis of protein. G.'s disintegration begins in the erythrocytes finishing the life cycle. Gem is oxidized on an alpha metinovomu to the bridge with an opening of bond between the corresponding rings of a pirrol.
The received derivative G. is called verdohemoglobin (a pigment of green color). It is very unstable and easily breaks up to an ion of iron (Fe 3+ ), the denatured globin and biliverdin.
The great value in G.'s catabolism is allocated for a gaptoglobin-gemoglobinovy complex (Hp — Hb). During the escaping of an erythrocyte in a blood channel of G. it is irreversible communicates with gaptoglobiny (see) in Hp — Hb a complex. After exhaustion of all number of HP in plasma G. it is absorbed by proximal tubules of kidneys. The ground mass of a globin breaks up in kidneys within 1 hour.
The catabolism gem in Hp — Hb a complex is carried out by reticuloendothelial cells of a liver, marrow and spleen with education bilious pigments (see). The iron which is chipping off at the same time very quickly comes to metabolic fund and is used in synthesis of new Molecules.
Methods of definition of concentration of hemoglobin. In a wedge, the practician G. determine by usually colorimetric method by a Sahli hemoglobinometer, based on measurement of amount of the hemin which is formed of G. (see. Gemoglobinometriya ). However depending on the content in blood of bilirubin and a methemoglobin, and also at some patol, states the error of a method reaches +30%. More exact are spectrophotometric methods of a research (see. Spektrofotometriya ).
For definition of the general hemoglobin in blood use the tsianmetgemoglobinovy method based on all derivative G.' transformation (dezoksi-Hb, HbO 2 , HbCO, met-Hb, etc.) in cyan - met-Hb and measurement of size of optical density of solution at 540 nanometers. For the same purpose use pyridine - a haemo chromogenic method. Concentration of HbO 2 usually determine by light absorption at 542 nanometers or a gas-metric method (by amount of the connected oxygen).
Hemoglobin in clinical practice
Determination of quantitative content and qualitative structure of G. is used in a complex with other gematol. indicators (an indicator of a hematocrit, number of erythrocytes, their morphology, etc.) for diagnosis of a row patol, conditions of red blood (anemia, erythremias and secondary hyperglobulias, assessment of degree of blood loss, a pachemia at dehydration of an organism and burns, etc.), for assessment of efficiency of hemotransfusions in the course of therapy etc.
Normal G.'s maintenance in blood averages for men 14,5 + 0,06 g of % (fluctuation of 13,0 — 16,0 g of %) and for women 12,9 + 0,07 g of % (12,0 — 14,0 g of %), according to L. E. Yarustovskaya and soavt. (1969); fluctuations depend on age and constitutional features of an organism, physical. activities, character of food, climate, partial pressure of oxygen in an ambient air. G.'s concentration in blood is relative size, depending not only from the absolute number of the general G. in blood, but also from the volume of plasma. Increase in volume of plasma at the invariable number of G. in blood can give the underestimated figures at G.'s definition and imitate anemia.
Apply also indirect indicators to more omnibus estimate of maintenance of G.: definition of a color indicator, average content of G. in one erythrocyte, srednekletochny concentration of G. in relation to an indicator of a hematocrit etc.
The decrease in concentration of G. which is found at severe forms of anemia in blood up to the certain critical size — 2 — 3 g of % and below (a gemoglobinopeniya, an oligokhromemiya) — usually conducts by death, however at some types hron, anemias certain patients owing to development of compensatory mechanisms adapt also to such concentration.
At patol, states G.'s maintenance and quantity of erythrocytes not always change in parallel that finds reflection in classification of anemias (distinguish normo-, hypo - and hyperchromic forms of anemia); the erythremia and secondary hyperglobulias are characterized by G. (giperkhromemiya) and increase in quantity of erythrocytes raised by concentration at the same time.
Practically all G. of blood is in erythrocytes; a part it is in plasma in the form of a complex of HP — Hb. Free G. of plasma makes normal 0,02 — 2,5 mg of % (according to G. V. Derviz and N. K. Byalko). Free G.'s maintenance in plasma increases at some hemolitic anemias proceeding preferential with an intravascular hemolysis (see. Haemoglobinaemia ).
Due to the existence of several normal types G., and also emergence in blood at some diseases of abnormal haemo globins of various origin (see. Hemoglobinopathies ) much attention is paid to definition of qualitative structure of G. of erythrocytes («a gemoglobinovy formula»). So, detection of the raised G.'s number like HbF and HbA2 is characteristic usually of some forms of a beta talassemia.
Increase in maintenance of HbF is noted also at other gematol. diseases (an acute leukosis, aplastic anemia, a paroxysmal night Haemoglobinuria, etc.), and also at infectious hepatitis, at an asymptomatic hereditary persistirovaniye of fetalis hemoglobin and pregnancy. Concentration of HbA2 fraction in blood increases in the presence of some unstable G., intoxications and decreases at an iron deficiency anemia.
In ontogenesis at the person change of various types of normal G. U of a fruit is noted (to 18 weeks) find primary, or primitive, hemoglobin P (English primitive); its versions designate the same as Hb Gower1 and Hb Gower2.
Primary G.'s dominance corresponds to the period of a vitelline hemopoiesis, and during the period of a hepatic hemopoiesis following it HbF is synthesized already preferential.
Synthesis of «adult» HbA is sharply intensified during marrowy blood formation; the maintenance of HbF at the newborn child makes up to 70 — 90% of total quantity of G. (other 10 — 30% fall on HbA fraction). By the end of the first year of life concentration of HbF usually decreases to 1 — 2%, and the maintenance of HbA respectively increases.
It is known St. 200 abnormal (patol. or unusual) options G. which emergence is caused by various hereditary defects of formation of polypeptide chains of a globin.
L. Polinga's opening, Itano (N. A. Itano) and sotr. in 1949 patol, hemoglobin S (English sickle cell drepanocytic) laid the foundation for the doctrine about molecular diseases. Existence in abnormal G.'s erythrocytes usually (but not always) leads to development of a syndrome of hereditary hemolitic anemia (see).
The majority of the described options of hemoglobin should be considered not pathological, but rather rare unusual Forms. From medical positions haemo globins of S, S, D, E, Bart, H, M and big group (apprx. 60) unstable G have a certain value. Unstable G. name abnormal options G. which have one replacements from amino acids instability of a molecule to action of oxidizers, heating and some other factors resulted. M-groups arise owing to replacements of amino acids in polypeptide chains in the field of contacts gem and a globin that results not only in instability of a molecule, but also in the increased tendency to a metgemoglobinoobrazovaniye. The M-hemoglobinopathy quite often is the reason hereditary methemoglobinemias (see).
G.'s classification was originally based on their image as opening by letters of the Latin alphabet; the exception is made for the normal «adult» G. designated by a letter A, and G. of a fruit (HbF). The letter S designated abnormal drepanocytic G. (a synonym of HbB). Thus, letters of the Latin alphabet from And to S were considered as the conventional Designations. It agrees accepted on X International gematol. the congress (Stockholm, 1964) G.'s nomenclature for designation of new options is not recommended to use from now on other letters of the alphabet.
Now it is accepted to call again opened forms G. in the place of opening with use of the name of the city (area),-tsy or laboratories where new G. was for the first time found, and with the instruction (in brackets) its biochemical, formulas, the place and the nature of amino-acid replacement in the struck chain. E.g., Hb Koln (alpha 2 beta 2 98 >val — met ) means that in hemoglobin Cologne there was a replacement in the 98th position of one of beta and polypeptide chains of amino acid of valine by methionine.
All kinds of G. differ from each other on physical. - chemical and physical. to properties, and some and on functional properties what methods of detection of various options G. in clinic are based on. The new class of abnormal G. with the changed affinity to oxygen is open. G.'s typing is made by means of an electrophoresis and some other laboratory methods (test on alkali fastness and a thermal denaturation, a spektrofotometriya, etc.).
Are divided by electrophoretic mobility of G. on fast-moving, slow and normal (having the mobility identical with HbA). However replacement of the amino-acid remains not always leads to change of a charge of a molecule G. therefore some options cannot be revealed by means of an electrophoresis.
Hemoglobin in the medicolegal relation
G. and its derivatives in forensic medicine are defined for establishment of availability of blood on material evidences or in any liquids at diagnosis of poisonings with the substances causing G.'s changes for difference of the blood belonging to a fruit or the newborn from blood of the adult. There are data on use of features of G. which are descended in examination of a doubtful paternity, motherhood and replacement of children, and also for individualization of blood on material evidences.
By immunization of animals hemoglobin of the person received gemoglobinpretsipitiruyushchy serums. By means of these serums in the spot investigated on G. presence of blood of the person can be established.
At establishment of availability of blood in spots microspectral analysis and microcrystallic reactions is applied. In the first case of G. by alkali and a reducer it is transferred to hemochromogen which has a characteristic absorption spectrum (see. Hemochromogen ), or affect G. with the concentrated chamois to - that that leads to formation of haematoporphyrin., the Last possesses a typical absorption spectrum in a visible part of a range.
From microcrystallic reactions for establishment of availability of blood most often use the tests based on receiving crystals of hemochromogen and muriatic hemin. For receiving crystals of hemin from fabric with the spot investigated on G. take a thread and place on a slide plate, add several crystals of sodium chloride and several drops concentrated acetic to - you (Teykhmann's reactant). During the heating (in case of presence of blood) crystals of muriatic hemin (Teykhmann's crystals) — brown color slanting parallelograms are formed of G., reactions of receiving from G. of crystals iodine-hemin — small crystals of black color in the form of rhombic prisms are sometimes applied.
Derivative G. spectroscopically are established in blood at some poisonings. E.g., at poisoning with carbon monoxide in blood of victims carboxyhaemoglobin is found, at poisoning with metgemoglobinobrazuyushchy substances — a methemoglobin.
In cases of infanticide happens necessary to establish presence of blood of the newborn or a fruit on various material evidences. As in blood of a fruit and the newborn the high content of HbF, and in blood of the adult — HBA distinguished on physical is observed. - to chemical properties, G. of newborn (fruit) and the adult can be easy otdifferentsirovana.
In practice most often use an alkaline denaturation since G. of a fruit is steadier against effect of alkalis, than G. of the adult. G.'s changes are established spectroscopically, spektrofotometrichesk or photometric.
Synthesis of polypeptide chains of G. is carried out under control of structural and (perhaps) regulatory genes. Structural genes cause a certain amino-acid sequence of polypeptide chains, regulatory — the speed of their synthesis (see. Gene ).
The existing 6 types of chains of normal (Hbα, Hbβ, Hbγ, Hbδ, Hbε, Hbζ) at the person are coded by respectively 6 gene loci (α, β, γ, δ, ε, ζ). Believe that for α-chains there can be two loci. Besides, 5 different γ-chains which are coded by different loci are revealed. Thus, all the person can have from 7 to 10 couples of structural genes controlling synthesis of.
Studying of stages of development showed that the person has accurate and well balanced genetic regulation of synthesis of various G. V to the first half of uterine life at the person hl are active. obr. loci α, γ, ζ, ε-chains (the last it is only short-term, in the early period of embryonal life). After the birth along with switching off of a locus of gamma chains loci β, δ-chains are activated. Such switching is resulted by fetalis HbF replacement by haemo globins of the adult — HbA with small fraction HbA2.
Remain not clear questions an arrangement of the gene loci defining G.'s synthesis on chromosomes, their coupling, dependence of the specific and connected with the periods of ontogenesis activation and repression of structural genes of G. from action of regulatory genes, influences of humoral factors (e.g., hormones) etc.
Synthesis of chains of a globin represents a private example of synthesis of protein in a cell.
Though in regulation of synthesis of G. there is a lot more not clear, however, apparently, the mechanisms controlling the speed of a transcription of IRNK (information RNA) with DNA are key. The exact characteristic of DNA specifically responsible for synthesis of a globin, is not received. However in 1972 at the same time in several laboratories it was succeeded to synthesize the gene regulating G. Eto's synthesis it was made by means of enzyme of the return transcriptase (see. Genetic engineering).
Gemovy part of a molecule G. is synthesized separately by means of a series of enzymatic reactions, since active succinate (amber to - you) from a tricarbonic acid cycle and finishing a difficult protoporphyrinic ring with atom of iron in the center.
In the course of proteinaceous synthesis globinovy chains obtain a configuration, characteristic of them, and gems «invests» in a special pocket. Further there is a combination of complete chains of G. to formation of tetramer.
Synthesis of specific DNA happens in predecessors of erythrocytes only to a stage of an ortokhromny normoblast. For this period a final set of polypeptide chains of a globin is created, there is its connection with gemy, all versions RNA and necessary enzymes are formed.
Inherited disorders of synthesis of G. are divided into two big groups:
1) so-called structural options or anomalies of primary structure of G. — «qualitative» hemoglobinopathies like Hb, S, S, D, E, M, and also the diseases caused by unstable G. and G. with the increased affinity to O 2 (see. Hemoglobinopathies ),
2) the states arising owing to the broken speed of synthesis of one of polypeptide chains of a globin — «quantitative» hemoglobinopathies or thalassemias (see).
At structural options stability and function of a molecule can change. At thalassemias the structure of a globin can be normal. Since both types of genetic defect are widespread in many populations of people, individuals, at the same time heterozygous by structural option G. and on a thalassemia are quite often observed. Combinations of various genes make very difficult range of hemoglobinopathies. In certain cases mutations can strike mechanisms of switching of synthesis of G. that leads, e.g., to continuation of synthesis of fetalis G. at adults. These states carry the group name of a hereditary persistention of fetalis hemoglobin.
With the merged chains mutants like Hb Lepore, anti-Lepore and Kenya belong to options. It is the most probable that these structural anomalies of G. arose owing to an unequal nonhomologous meiotic crossing-over between closely linked genes of. As a result of it, e.g., in Hb Lepore of a α-chain are normal, and other polypeptide chains contain a part of the sequence δ-and a part of the sequence of β-polypeptide chains.
As mutations can arise in any of the genes defining G.'s synthesis there can be several situations at which individuals will be homozygotes, heterozygotes or double heterozygotes on abnormal G.' alleles in one or several loci.
More than 200 structural options G. are known, from them more than 120 are characterized, and in many cases it was succeeded to connect structural change of G. with its abnormal function. The simplest origins of new option G. as a result of point mutation (replacement of the only basis in a genetic code) can be shown on the example of HbS (scheme).
Influence of amino-acid substitution with physical. - chemical properties, stability and function of a molecule G. depends on type of amino acid, replaced edges former, and its provisions in a molecule. A number of mutations (but not all) significantly change function and stability of a molecule G. (HbM, unstable haemo globins, haemo globins with the changed affinity to O 2 ) or its configuration and row physical. - chemical properties (HbS and HbC).
Haemo globins unstable
Gemoglobina unstable — group of the abnormal haemo globins differing in special sensitivity to action of oxidizers, heating and some other factors that is explained by genetically determined replacement in their molecules of one amino-acid remains by others; the carriage of such haemo globins is often shown as hemoglobinopathy (see).
In erythrocytes of people — unstable G.' carriers there are so-called little bodies of Heinz representing accumulations of the denatured unstable G.'s molecules (inborn hemolitic anemia with Heinz's little bodies). In 1952. I. A. Cathie suggested about the hereditary nature of this disease. To P. Frick, W. H. Hitzig and the Branch (To. Betke) in 1962 for the first time on the example of Hb Zurich proved that hemolitic anemia with Heinz's little bodies is connected with presence of unstable haemo globins. R. W. Carrell and G. Lehmann in 1969 offered the new name of such hemoglobinopathies —
can be called the hemolitic anemias caused by unstable G. Nestabilnost's carriage of molecules G. by replacement of the amino-acid remains contacting with gemy; replacement of the rest of unpolar amino acid by polar; disturbance of secondary structure of the molecule caused by replacement of any amino-acid rest with the rest of proline; replacement of the amino-acid remains in the area α1β1-and α2β2-контактов that can lead to dissociation of a molecule of hemoglobin on monomers and dimeasures; deletion (loss) of some amino-acid remains; lengthening of subunits, napr, two unstable G. — Hb Cranston and Hb Tak have the beta chains extended in comparison with normal hemoglobin at the expense of the hydrophobic segment attached to their C-end.
The unstable G.' classification offered J. V. Dacie and modified Yu. N. Tokarev and V. M. Belostotsky, it is based on the nature of the changes in a molecule doing G. unstable.
It is described apprx. 90 unstable G., and options in beta chains of a molecule G. meet replacement of the amino-acid remains approximately by 4 times more often than with replacement of such remains in alpha chains.
Unstable G.' carriage is inherited on autosomal dominantly type, and carriers are heterozygotes. In some cases emergence of a carriage of unstable G. is result of natural mutation. Decrease in stability of G. not only is led to its easy precipitation, but in some cases and to loss by gem. Substitutions of the amino-acid remains in places of contacts and - and (3 chains of a molecule G. can influence affinity of a molecule to oxygen, interaction of gem and balance between tetramers, dimeasures and monomers of hemoglobin. At people, heterozygous on unstable G.'s genes, both normal, and abnormal, unstable G. is synthesized, however the last quickly denatures and becomes functionally inactive.
Heavy hemolitic anemia is usually noted at patients, being unstable G.' carriers with high degree of instability of a molecule.
At other unstable G.' carriage the wedge, manifestations usually happen moderately severe or absolutely insignificant. In some cases (Hb Riverdale — Bronx, Hb Zurich, etc.) unstable G.'s carriage is shown in the form of hemolitic crises after reception of some drugs (streptocides, analgetics, etc.) or influences of infections. At some patients, napr, carriers Hb Hammersmith, Hb Bristol, Hb Sydney, etc., the cyanosis of skin caused by the increased education met-and sulfhemoglobins is noted. The hemoglobinopathies caused by unstable G.' carriage should be differentiated with hemolitic and hypochromia anemias of other etiology and first of all with the iron deficiency and hemolitic anemias connected with genetically caused deficit of enzymes of a pentozo-phosphatic cycle, glycolysis, etc.
Most of people — unstable G.' carriers does not need special treatment. At hemolysis fortifying therapy is useful. All carriers of unstable G. are recommended to abstain from the drugs oxidizers provoking hemolysis (streptocides, sulphones, analgetics, etc.). Hemotransfusions are shown only at development of deep anemia. At heavy hemolysis with the spleen raised by sequestration of erythrocytes and a hypersplenism it is shown splenectomy (see). However the splenectomy to children (up to 6 years) is usually not made because of risk of development of a septicaemia.
Methods of identification of unstable haemo globins
the Research of thermolability of hemoglobin — the most important test of detection of its instability. It is offered by A. G. Grimes and A. Meisler in 1962 and Deysi in 1964 and consists in an incubation of hemolysates, the diluted 0,1 M phosphatic or tris-HCl the buffer, pH 7,4, at 50 — 60 ° within an hour. At the same time unstable G. are denatured and drop out in a deposit, and the number of the thermostable G. which remained in solution is defined by spektrofotometrichesk at 541 nanometers and calculate by a formula:
[E of pilot test] / [E of check] * 100 == thermostable hemoglobin (as a percentage),
where E — the size of an extinction at the wavelength of 541 nanometer.
Abundance of thermolabile G. is equal to 100% — thermostable G.'s number (as a percentage).
Karrell y Kay (R. Kau) in 1972 hemolysates in mix of 17% solution of isopropanol — the tris-buffer, pH 7,4 suggested to incubate at 37 ° within 30 min.
Hemolysis of erythrocytes can be caused water since use for this purpose of perchloromethane or chloroform leads to a partial denaturation of unstable G. and distortion of the obtained data.
The most widespread method of definition of unstable G. is gistokhy, the method of identification of little bodies of Heinz. Erythrocytes at the same time paint crystal violet, methyl violet or use reaction with acetylphenylhydrazine. Blood is previously maintained within a day at 37 °. It must be kept in mind that Heinz's little bodies can be found also at other hemolitic anemias, a thalassemia, at poisoning with methemoglobin formers and at some enzymopathies.
Electrophoretic division of hemolysates on paper or acetate-cellulose often does not yield results since replacement of the amino-acid remains in a molecule does not cause change of electrophoretic properties of a molecule in many unstable G. Are more informative in this respect an electrophoresis in poliakrilamidny and starched gels (see. Electrophoresis ) or isoelectric focusing.
At many patients, being unstable G.' carriers, urine constantly or times gains dark color owing to formation of dipirrol that is rather exact sign of presence at erythrocytes of unstable G.
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A. P. Andreyeva; BB. H. Tokarev (gems. and gen.), A. K. Tumanov (court.).; BB. H. Tokarev, V. M. Belostotsky.