GLYCOPROTEINS (glycoproteins) — the biopolymers consisting from covalently the peptide (proteinaceous) and carbohydrate components connected among themselves. When a carbohydrate part of a molecule G. consists of a glucose unit or the remains, G. carry the name of gluco-proteids. Are found in an organism of animals, bacteria and plants and make the most extensive and well studied class of uglevodsoderzhashchy connections. Are a part of a cellular cover, circulate in a blood channel as transport molecules — napr, transferrin, ceruloplasmin (see. Blood ). Some hormones, enzymes, and also immunoglobulins belong to glycoproteins. One of the first assumptions for what proteins need a carbohydrate component, was made by Eylar (E. H. Eylar, 1965). By consideration of quite large number of G. he found out that all of them are out of a cell, in a blood channel, saliva, milk and other secrets. On the basis of this fact it assumed a hypothesis, according to a cut the carbohydrate component is some kind of admission, after receiving to-rogo a proteinaceous molecule shall leave a cell surely. Further, however, the data testimonial of the fact that many intracellular proteins are G. were obtained and are a part of various intracellular membranes and a cytoplasmic membrane. Besides, in various secrets and blood serum neglikozilirovanny proteins were found (albumine, a-lactalbumin, chymotrypsinogen, etc.). Thus, Eylar's hypothesis cannot apply for universal permission of a question of a role of a carbohydrate component. In further researches it was revealed that if from a carbohydrate part of a number of serumal G. (ceruloplasmin, gaptoglobin, feta in, orozomukoid) to chip off enzymatically sialine to - that, then time of half-decay of these asialo-glycoproteins will be reduced of several tens hours to several minutes. At the same time all specified asialo-glycoproteins contact membranes of parenchymatous cells of a liver.
A number of glycoproteins - hormones (e.g., gonadotropic and follicle-stimulating hormones) after removal sialine to - t very quickly disappears from a blood-groove and, as well as serumal G., it appears in hepatic cells. As a result hormone does not contact target cells and it biol, action sharply decreases.
Sialic acid (see) can define, apparently, time of life in a blood-groove not only molecules G., but also some cells, in particular erythrocytes. Its eliminating from a membrane of erythrocytes leads to their bystry disappearance from a blood-groove.
Except performance of informative function (where to go to molecules or cells), carbohydrate chains of G. increase stability of molecules which part they are, and protect them from effect of proteolytic enzymes. Many glycoproteins enzymes, in particular lysosomic a glycoside elements, have surprising stability, keeping completely the activity in homogenates at t ° 37 ° during several tens hours. In model experiences it was established that after accession of a carbohydrate part to a nek-eye to enzymes — alpha and to beta amylases, to trypsin, and also to a nek-eye to proteins (concanavalin A) their heat stability and proteolysis resistance substantially increases.
One more Property is recently revealed. It turned out that in blood and tissues of the Antarctic fishes there are glycoproteins antifreezes interfering formation of crystals of ice in an organism of fishes at t ° there is lower than 0 °.
, contained in various slizyakh of inner and outer surfaces of an animal organism, carry out a protective role at action of various factors internal and the environment and participate in protection of an organism against penetration of bacteria.
G.'s role in intercellular interactions is one of the most intensively developing directions in their research. Being a part of a cellular cover, G. play an important role in ion exchange of a cell," immunological reactions, a differentiation of fabrics, the phenomena of intercellular adhesion etc.
The size of molecules G. and a ratio of carbohydrate and proteinaceous components vary over a wide range. G. about a pier are known. (ribonuclease B) weighing from 15 000 and to 1 000 000 and above (G. from saliva of the person and animals). The carbohydrate component can make from 1 — 3% (ovalbumin, various kollagena) to 80 — 90% (group substances of blood) of all Molecule.
The number of the carbohydrate chains attached to peptide «core» of G. can be very various. In some G. (ribonuclease B., transferrin, an acid alfa1-glycoprotein) the number of carbohydrate chains does not exceed 1 — 4, in other types G. (group substances of blood, some mucins from submaxillary glands) the number of carbohydrate chains reaches 300 — 800. It should be noted high heterogeneity of a carbohydrate Component. Even in different molecules G. of one type allocated from the same source distinctions in number and the nature of carbohydrate links in a chain and chains, and also in type of a glycosidic linkage between the carbohydrate remains are observed. Differ in a big variety of forms of the molecules: from globular (ovalbumin) to strongly extended, characteristic, e.g., for G. from submaxillary glands of the person and animals.
Monosakharidny components G. are hexoses (see) — a D-galactose, D-mannose, D-glucose, pentoses (see) — D-xylose and L-pectine sugar and aminosugar (see) — N-atsetilglyukozamin and N-acetyl galaktozamin in which the amino group at the second carbon atom is usually acetylated. Representatives of deoxysugars — L-fukoza and L-rhamnose are G.'s part. A typical component G. is neuraminic (sialine) to - that.
Peptide part G. is characterized by certain features which establishment allowed to clear up G.'s classification and to subdivide them into groups on the basis of types carbohydrate peptide bonds.
The glycoside amide bonding between N-atsetilglyukozaminom and beta amide nitrogen can be an example of such bonds asparagine (see). This communication is rather steady against action to - t and alkalis and often meets in G. of a blood plasma, immunoglobulins, glycoproteins enzymes, glycoproteins hormones. Peptide part G. with glikozilamidny bonding is characterized by the ordinary amino-acid structure typical for the majority of proteins.
O-Glikozidny bonding is noted during the binding of a glikozidny part with serine (see) or threonine (see). N-acetyl galaktozamin (mucins, group substances of blood), or the galactose (e.g., G. of a cuticle of an earthworm) can be the sugar participating in formation of this bonding or. The peptide part G. which is characterized by this bonding differs in the high content of serine and threonine, components sometimes (in group substances of blood) apprx. 50% of all amino acids. Glycoproteins of this group contain also significant amount of proline (from 11 to 20%).
O-Glikozidny bonding with oxylysine or oxyproline is subdivided into two look. Galaktozil-oksilizinovy communication is characteristic of collagen. The second type of communication — arabinozil-oksiprolinovy communication is available in G. of the higher plants.
Biosynthesis of carbohydrate chains of G. is not matrix, and is carried out by means of several glikoziltransferaz specific both concerning donors of the glikozilny remains, and concerning their acceptors. The nukleozidfosfatsakhara and so-called lipidic carriers of which 20 century became known only at the beginning of the 70th usually act as donors of the glikozilny remains. A lipophilic part of molecules of these connections represents the polynonlimiting alcohol constructed of the remains of an isoprene, to Krom through one (poliprenolmo-nofosfatsakhara) or two (poliprenolipirofosfatsakhar) of the rest phosphoric to - you are attached the carbohydrate rest.
Initiation of biosynthesis of carbohydrate chains of G. begins after biosynthesis on ribosomes of a polypeptide chain by means of various glikoziltransferaz catalyzing transfer of the first monosakharidny rest from the donors stated above. It also defines type carbohydrate peptide bond. Further accession of the carbohydrate remains, i.e. lengthening of a chain — elongation, and the last stage — termination also happens with the participation of various glikoziltransferaz which are carrying out consecutive addition of monosakharidny links.
Glikoziltransferaza form family, each of which carries out transfer only of one type of monosaccharides. So, families fukoziltransferaz, sialiltransferaz, galaktoziltransferaz, etc. are found. Each member of such family, attaching the same monosakharidny rest, can have absolutely various specificity in relation to a pier. to the weight of a molecule of an acceptor, its conformation and other properties, and also in relation to the nature of trailer, directly glikoziliruyemy rest or even the monosakharidny rest attached to the trailer rest. Glikoziltransferaza of one family can differ in two features: a) they can need specific molecules of an acceptor, b) at a glycosylation of the same acceptor molecule of transferase can attach the monosakharidny rest with formation of various types of a glycosidic linkage, synthesizing thereby various end products of biosynthetic reaction. All members of one family glikoziltransferaz need the same nukleoziddifosfatsakhara as donors of the glikozilny remains.
With participation in G.'s biosynthesis of lipidic carriers of reaction of a glycosylation can proceed on an interface between a lipidic and aqueous phase biol, membranes. To acceptor molecules in these cases not only the monosakharidny remains, but also oligosakharidny chains can be moved. It is supposed that lipidic carriers participate in biosynthesis of internal carbohydrate circuits of G. which are characterized a glycoside by amide type carbohydrate peptide bond.
G.'s variety is defined by type of fabrics in which usually activity of members of one family glikoziltransferaz is rather high, and others — is reduced or completely is absent. See also Gaptoglobin , Glycosides , Group-specific substances , Blood groups .
Biochemical methods of definition of glycoproteins
In biol, liquids, blood and urine there is a mix of various G. Allocation of each of them in pure form requires a difficult technique, a long time, as complicates use it for serial researches. Therefore in a wedge, practice total definition of G. is most widespread on one of the components of a carbohydrate part entering them — hexoses, hexosemines, a fukoza, sialine to - there or on ability to give reaction iodic to - that is Schiff's reactant (see. Schiff reactant ).
The most applied methods of definition of G. can be divided into two basic groups conditionally: chemical and electrophoretic. Hromatografichesky, polyarografichesky and radio immunological methods are applied to special researches.
The majority of chemical methods is based on definition of a carbohydrate part of a molecule G. using various staining reactions based on interaction of a monosaccharide with a chamois to - that with formation of derivative furfural (e.g., reactions with ortsiny, Anteronum, tryptophane, a carbazole, biphenyl amine, resorcin, alpha-naphthol). Such reactions give the painted product with one of the listed connections or aromatic nitrogen base. The quantity of the formed painted product is defined by a fotoelektrokolorimetrirovaniye. The method of definition of hexoses using staining reaction with ortsiny or resorcin is considered the most exact; the most sensitive — a method using alpha-naphthol which, however, is more often used for approximate researches.
Almost all methods applied to definition of aminosakharid are based on a classical method of Elson — Morgan (1933). The principle of a method is that aminosugar reacts about acetyl acetone in hot alkalescent solution. At the same time the mix of pirrol giving red coloring with reagent para-dimethylaminobenzaldehyde is formed, intensity to-rogo is defined photometric at 530 nanometers. The glycosamine gives almost same coloring, as well as galaktozamin; with mannosamine coloring is slightly weaker. For creation of a standard curve generally use a muriatic glycosamine.
Reaction is applied to definition of a fukoza, in a cut to a product of interaction of G. with a chamois to - that muriatic cysteine increases.
This reaction is a basis of almost all methods applied to definition of methyl pentoses (see. Dishe method ).
For detection and quantitative definition sialine to - a number of methods is offered t: an ortsinovy method with Bial's reactant, a resorcinol method, a method with thiobarbituric to - that, difenilaminovy reaction and Hess's method (see. Hess reaction ). The most sensitive and specific is the method with thiobarbituric to - that. G.'s electrophoresis was introduced for the first time Keivy Ridge and Gryonvall (E. Koiw, A. Gronwall) in 1952. A number of modifications of this method is described; common fault of most of them is comparative complexity of a method or considerable coloring of a background on elektroforegramma. The principle of a method and technology of performance of an electrophoresis of, the same, as at electrophoretic division of protein fractions of blood serum on paper (see. Electrophoresis ).
For identification of glikoproteidny fractions a number of methods is offered; coloring by toluidine blue, colloid iron, antsianovy blue, etc. However the method of coloring of glycoproteins is most extended by Schiff's reactant, the basis to-rogo was formed by the reaction (iodic to - that is fuksinsernisty to - that) which is originally offered by R. D. Hotchkiss and J. F. A. McManus for G.'s coloring in gistol, cuts. The principle of this method consists that carbohydrate components G. are oxidized solution iodic to - you to aldehydes, and aldehydes come to light by means of Schiff's reactant. For quantitative definition the painted fractions are eluated with elektroforegramm with the subsequent fotometrirovaniye of eluates or decide on the help densitometries (see).
At the healthy person, according to various authors, abundance of fractions G. (in %) the following: albuminous — 10,4 — 16,6; alpha 1 - globulinovy — 14,2 — 18,3; alpha 2 - globulinovy — 24,8 — 31,8; beta globulinovaya — 21,7 — 25,0; uglobulinovy — 16,0 — 19,2.
The highest percent of content of carbohydrates is noted in globulinovy fractions, in particular in alfa2-and beta globulins (see. Globulins ).
The method is perspective immunoelectrophoresis (see), and also a method of an electrophoresis in polyacrylamide gel (PAAG), resolving power to-rogo can exceed resolving power of paper electrophoresis by 10 times.
Results of definition of glycoproteins have important differential and diagnostic value at diseases of connecting fabric, cardiovascular system, went. - kish. path, liver, kidneys, lungs. At the same time studying of changes in the content of these substances in blood serum and others biol, liquids in addition to a wedge, to data is of great importance for assessment of a current patol, process, efficiency of treatment and for the forecast. At inflammatory processes — acute rheumatism, tuberculosis, pneumonia, pleurisy increase in content in blood serum of all fractions G., especially an alpha is noted 1 - and alpha 2 - globulins. The greatest value has definition absolute and abundance of G. in blood serum at rheumatism. G.'s concentration in blood serum increases also at a glomerulonephritis, tumors, a necrosis, is frequent at diabetes.
Histochemical methods of definition of glycoproteins in fabrics
Gistokhim, methods of detection of G. are based on identification of their reactive groups, such as 1,2-glikolny groups, and also carboxyl groups sialine to - t. For G.'s fixing it is possible to use 10% solution of formalin at t ° from 0 to 4 ° within 24 — 48 hour. There are methods of fixing with addition in formalin of some salts, and also various cationic detergents (see), the polysaccharides promoting the best safety and their the subsequent gistokhy, a differentiation. Preference should be given to a method of freeze drying of cuts with the subsequent paraffin embedding. There is a considerable number gistokhy, methods and their modifications of identification of polysaccharides, but not everyone they is sufficiently reliable and available in the practical relation. The most reliable at obligatory use of control reactions and proved from the point of view of chemistry is the method of IAC Manus — Hochkissa — Shabadasha. In laboratories of our country Shabadash's modification is most often applied. Oxidation of 1,2-glikolny groups of polysaccharides salt iodic to - you with the subsequent identification of the aldehydes received as a result of reaction fuksinsernisty to - that is the cornerstone of a method (Schiff's reactant). In sites of localization muko-and glycoproteins violet-red coloring of various intensity develops. Except these connections, reaction reveals also a glycogen, glycolipids and free aldehydes. Nonspecific aldehydes can appear also as a result of oxidation of connections with unsaturated bonds during fixing of material formalin. Therefore control is necessary for obtaining reliable results careful gistokhy: first of all it is necessary to exclude availability of free and nonspecific aldehydes and if they are present, to carry out reaction of their blocking. Applying a malt diastase (as a last resort ptyalin), it is possible to exclude existence of a glycogen.
Necessary reactants: crystal main fuchsin (or so-called main fuchsin for fuksinsernisty to - you), 1 N of HCl, potassium metabisulphite or sodium (K 2 S 2 O 5 or Na 2 S 2 O 5 ), peryodny to - that or is better than it potassium salt (KIO 4 ), high cleaning malt diastase, muriatic hydroxylamine. For reaction of acetylation: anhydrous pyridine and acetic anhydride, 0,1 N caustic heat (KOH), drug of a neuraminidase. Lipids delete with processing by various solvents (e.g., hot mix of chloroform and methyl alcohol).
Course of definition. Serial sections, experienced and control, are exposed to processing by solution of periodate of potassium, quickly wash out in a dist, to water and place in Schiff's reactant, then wash out in freshly cooked solution of bisulphite (10 ml of 10% of solution of potassium metabisulphite, 10 ml 1 N of HCl and 200 ml of the diart. of water). Cuts carefully wash in the large volume of water, dehydrate in alcohols, clarify in a xylol and conclude in neutral Canada balsam.
Reaction of acetylation of glikolny groups and use of a neuraminidase confirm reliability of the received results.
Bibliography Anasashvili A. Ts. Glycoproteins of blood serum and urine, M., 1968, bibliogr.; Vidershchayn G. Ya. Uglevodsoderzhashchiye of connection, their biosynthesis and a role in a zooblast, Mo lek. biol., t. 10, No. 5, page 957, 1976, bibliogr.; Glycoproteins, under the editorship of A. Gottschalk * the lane with English, t. 1 — 2, M., 1969; D ere-vitsky V. A. Himiya of glycoproteins, Usp. biol. chemical under the editorship of B. N. Stepanenko, t. 8, page 168, M., 1967; Methodical instructions on use of the unified clinical laboratory methods of researches, under the editorship of V. V. Menshikov, M., 1973; Pearce E. A histochemistry, the lane with English, page 741, etc., M., 1962; The Principles and methods of gisto-cytochemical analysis in pathology, under the editorship of. And. Item Avtsy-na, etc., page 7, L., 1971; Spiro R. G. Glycoproteins, Advanc. Protein Chem., V. 27, p. 349, 1973, bibliogr.
G. Ya. Vidershayn; A. Ts. Anasashvili (mt. issl.), P. A. Simakova (gist.).