[Effect of polysaccharides on biological activity of human lactoferrin].

The influence of neutral and ionic polysaccharides on the antioxidant (AOA) and detoxifying activities of lactoferrin (LF) and the duration of its circulation in the body was studied. In addition to natural polymers, we studied artificial chitosan derivatives with different functional groups. On the basis ofAOA test, five polysaccharides were selected. The study of the detoxifying effect of LF in two models of induced toxicity revealed polysaccharides that maintained or increased the detoxifying activity of LF. We established that the formation of a complex of lactoferrin with two galactomannans and succinyl chitosan caused positive changes in LF properties: the detoxifying activity of the protein remained unchanged or increased, whereas its elimination from the body was decelerated.

[Sulfated polysaccharides and their anticoagulant activity (review)].

Published data on the sulfated polysaccharides of various origins that display an anticoagulant activity are summarized and analyzed. The methods used for producing semisynthetic derivatives are considered. A key role of the polysaccharide structure in the mechanisms of specific interaction with various blood plasma proteinases is discussed. The effects of the content and location of sulfate groups in polysaccharides and their molecular weight on the degree of the studied activity are assessed.

[Effects of intravenous injection of sulphated galactomannan on anticoagulant activity of rat plasma].

We studied anticoagulant activity in vitro and in vivo of sulfate depolymerisation galactomannan guar with the following characteristics: Man:Gal 1.64, molecular mass 127 kDa, sulfation degree 1.46. We found the ability of galactomannan-HSO3Na (GM) to increase the time of blood coagulation in the test aPTT with an increase of its concentration and to decrease velocity of chromogenic substrate on the factor Xa hydrolysis. Specific antithrombin and anti-factor Xa activities of GM were 35.8 +/- 1.8 IU/mg and 6.6 +/- 0.5 IU/mg, respectively. In biospecific electrophoresis complexes arise between GM and protamine sulfate. Intravenous injection of GM to rats prolonged plasma coagulation time in the aPTT test with dose reduction from 1 to 3 mg/kg. In rat thrombosis model a dose of 3 Mg/kg produced a 100% inhibition of blood clot.

[Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds].

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3(-) groups was 48.05% +/- 2.31. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests--aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65-87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.

[Depolymerisation of legume seed galactomannans by Celloviridin G20x].

The depolymerisation of legume seed Galactomannans by commercial preparation "Celloviridin G20x" to produce fragments of macromolecules with unchanged monomer ratio was studied. The hydrolysis of four galactomannans in whole range of monomer ratio for this phytopolysaccharide group 1.1-5.1 was performed by enzyme preparation, which is a sum of carbohydrases (producent--Trichoderma reesei). Degree of substitution 1,4-beta-D-mannopyranose chain on C-6 position by alpha-galactose residues had influence on values of molecular mass of final reaction products. The fragments with high yelds and monomer ratio nearly of that initial polysaccharides were obtained.

[Synthesis of galactomannan sulfates].

The experimental conditions for the sulfation of legume galactomannans were found, which allow for the obtainment of polysaccharides with a high degree of substitution. Sulfate esters of four galactomannans of different composition (galactose content, 16.4-47.5%) were synthesized using the SO3-pyridine complex in dimethylformamide as a sulfating agent. The degree of substitution was as high as 1.4-1.8; it did not correlate with the content of galactose in the polysaccharides. It was found that the degree of sulfation depended on the reaction temperature in the range of 19-60 degrees C.

[Study of galactoglucomannans from the seed of Cercis canadensis L].

Galactoglucomannans were isolated by selective precipitation from aqueous and alkaline extracts of endosperm and hulls of Cercis canadensis, a member of the family Fabaceae. Their monosaccharide composition (Man: Gal: Glu) was as follows: 10.4: 0.9: 1 (polysaccharide from the endosperm) and 4.5: 0.9: 1 (polysaccharide from the hulls). The identity of IR spectra was indicative of the commonness of their structure. Analysis of the structure of the galactoglucomannan from endosperm by 13C NMR spectroscopy showed that its main chain consisted of 1,4-beta-D-manno- and 1,4-beta-D-glucopyranose. Part of mannose residues in the chain were substituted at C6 with single residues of alpha-D-galactopyranose. Galactoglucomannans are located in different part of the seed and implement different functions.

[Fractional isolation and study of the structure of galactomannan from sophora (Styphnolobium japonicum) seeds]. / Vydelenie galaktomannanov semian sofory iaponskoi (Styphnolobium japonicum) i izuchenie ikh struktury.

Two fractions (1 and 2) of the galactomannan from seeds of sophora (Styphnolobium japonicum) were isolated using cold and hot aqueous extraction with a total yield of 12.88%. The two fractions differed by the ratio between mannose (Man) and galactose (Gal) residues (4.8:1 and 5.3:1, respectively) and molecular weight (1190 and 1400 kDa, respectively). Aqueous solutions of these fractions were optically active ([alpha]D +4.80 degrees and -3.36 degrees, respectively) and highly viscous ([eta] 1028.8 and 1211.2 ml/g). 13C NMR spectra of both fractions were identical with respect to the number and positions of signals, which indicates that their primary structures were identical. Using chemical and spectroscopic (IR and NMR) methods, it was shown that the galactomannan has a main chain consisting of 1,4-beta-D-mannopyranose, some residues of which (16 and 17% in fractions 1 and 2, respectively) are alpha-galactosylated at the C-6 position. Frequencies of differently substituted mannobiose blocks in the chain, calculated for fraction 1 using NMR spectroscopic data, were 0.13 for the disubstitited blocks Gal(Man-Man)Gal, 0.37 for the sum of monosubstituted blocks Gal(Man-Man) and (Man-Man)Gal, and 0.50 for the unsubstituted block Man-Man.

[Composition and structure of galactomannan from the seed of Gleditsia ferox Desf]. / Sostav i struktura makromolekuly galaktomannana semian Gleditsia ferox.

Galactomannan, a heteropolysaccharide with a molecular weight of 1660 kDa, was isolated form the seed of Gleditsia ferox Desf., introduced in Russia, with a yield of 18.9%. Its aqueous solutions were optically active ([alpha]D = +30.5 degrees) and highly viscous ([eta] = 1430 ml/g). Analysis of the heteropolysaccharide using chemical, enzymatic, and chromatographic procedures showed that it consists of D-mannopyranose and D-galactopyranose residues (molar ratio, 2.54:1). The main chain of this galactomannan consists of 1,4-beta-D-mannopyranose residues, 39.2% of which are substituted at C6 with single residues of alpha-D-galactopyranose. The probability of occurrence of mannobiose units differentially substituted with galactose was determined by 13C-NMR data and equaled, respectively, 0.37, 0.47, and 0.16 for non-substituted Man-Man units, monosubstituted Gal(Man-Man) and (Man-Man)Gal units taken together, and for the disubstituted Gal(Man-Man)Gal units.

[Water-soluble galactomannan from the seeds of Lotus corniculatus L.: structure and properties]. / Vodorastvorimyi galaktomannan semian Lotus corniculatus L.: stroenie i svoistva.

Galactomannan, a water-soluble heteropolysaccharide, was isolated from the seed of Far-Eastern population of ground honeysuckle Lotus corniculatus L. (yield, 1.65%). Analysis of this galactomannan showed that is consists of D-mannose and D-galactose residues (molar ratio, 1.22:1). Its aqueous solutions were characterized by specific rotation [alpha]D = +84.10 and characteristic viscosity [eta] = 559 ml/g. Analysis of this heteropolysaccharide using chemical and enzymatic procedures, as well as IR- and 13C-NMR spectroscopy, showed that its main chain comprises 1,4-beta-D-mannopyranose residues, 95.5% of which are substituted at C-6 with single residues of alpha-D-galactopyranose.

[Determination of the primary and fine structures of galactomannan from seeds of Gleditsia triacanthos f. intermis L]. / Ustanovlenie perevichnoi i tonkoi struktury galaktomannan semian Gleditsia triacanthos f. inermis L.

This was the first study to isolate galactomannan, a 660-kDa polysaccharide, from the seeds of Gleditsia triacanthos f. inermis L. (yield 15.4%). Its aqueous solutions were optically active ([alpha] D = +31.0 degrees) and highly viscous ([eta] = 578 ml/g). The analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR- and 13C-NMR spectroscopy, showed that is consists of D-mannopyranose and D-galactopyranose residues (molar ratio 2.42:1). Its main chain is comprised of 1,4-beta-D-mannopyranose residues, 41% of which is substituted at C-6 with single residues of alpha-D-galactopyranose. The probability of occurrence of differently substituted mannobiose units in the chain, determined experimentally, was 0.16 for the unit Man-Man, 0.50 for the units Gal(Man-Man) and (Man-Man)Gal, and 0.34 for the dissubstitued unit Gal(Man-Man)Gal.

[Localization of acetyl groups in macromolecules of glucomannan from Eremurus zangezuricus roots]. / Lokalizatsiia atsitil'nykh grupp v makromolekule gliukomannana iz kornei Eremurus zangezuricus.

Water-soluble glucomannan from roots of Eremurus zangezuricus Mikheev was studied. The polysaccharide contains D-glucose, D-mannose, and acetyl groups in the molecular ratio of 1:2.8:0.38. 13C NMR studies showed that the polysaccharide under study is a linear, partly acetylated 1,4-beta-D-glucomannan. The acetyl groups are attached to the C2 and C3 of mannopyranose units. The polymer contains, on average, one acetyl group per seven mannose units. The glucomannan of E. zangezuricus has the following parameters: [alpha]D = -37.5 degrees, [eta] = 3.73 dl/g, and Mw = 151 kDa.

[Formation of xylitol in Candida guilliermondii 2581 culture]. / Obrazovanie ksilita v kul'ture Candida guilliermondii 2581.

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.

[Galactomannan from ambiguous Crazyweed (Oxytropis ambigua (Pall) DC)]. / Galaktomannan ostrolodochnika skhodnogo (Oxytrophis ambigua (Pall) DC).

A galactomannan with a molecular weight of 735 kDa was first isolated and purified from seeds of ambiguous crazyweed Oxytropis ambigua (Pall) DC. (family Leguminosae) with a yield of 3.6%. Its aqueous solutions displayed an optical activity ([alpha]D = 73.32 degrees) and high viscosity ([eta] = 644 ml g-1). Chemical analysis and 13C-NMR spectroscopy revealed the presence in the heteropolysaccharide of D-mannopyranose and D-glucopyranose at a molar ratio of 1.39:1. The linear backbone of its macromolecule consists of 1.4-beta-D-mannopyranose residues. Single beta-D-galactose residues substitute 72% of mannoses to form branches.

[Structure and characteristics of glucomannans from Eremurus iae and E.zangezuricus: detection of acetyl group localization in macromolecules]. / Struktura i khrakteristika gliukomannanov éremurusov Eremurus iae i E. zangezuricus, ustanovlenie lokalizatsii atsetil'nykh grupp v makromolekulakh.

Water-soluble glucomannans from roots of Eremurus iae and E. zangezuricus were studied. These polysaccharides were shown to contain 28.8; 69.0, and 2.2% (E. iae) and 22.6; 74.8, and 2.6% (E. zangezuricus) of D-glucose, D-mannose and acetyl groups, respectively. Their IR spectra were identical and revealed the presence of 1,4-beta-glycosidic bonds and ester carbonyl groups. 13C-NMR spectroscopy revealed both polysaccharides to be linear partially acetylated 1,4-beta-D-glucomannans. Acetyl groups substituted C-2- and C-3-hydroxyls of mannopyranose residues. Comparison of 13C-NMR data and the results of correlation analysis enables a conclusion to be made that acetyl groups can substitute no more than one OH-group in the mannopyranosyl residue. [alpha]D = -34.0 degrees, [eta] and molecular weights (MW) for E. iae polysaccharide were determined to be -34.0, 6.5 dl/g, and 265.5 kDa, respectively, and for E. zangezuricus polysaccharide -38.2, 5.4 dl/g, and 233.5 kDa, respectively. A correlation between intrinsic viscosities of polysaccharides and their molecular masses determined by HPLC was revealed.

[Composition and structure of a galactomannan macromolecules from seeds of Astragalus lehmannianus Bunge]. / Sostav i struktura makromolekuly galaktomannana semian Astragalus lehmannianus Bunge.

The composition and structure of a galactomannan from seeds of Astragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45% D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation, [alpha]D, equaled +81.3 degrees; the characteristic viscosity, [eta], 868.4 ml/g). Chemical, chromatographic, and spectral (IR and 13C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-beta-D-mannopyranose, 78% of which are substituted at position 6 with single alpha-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence, per pair of neighboring mannose units, of (1) two substituents, (2) one substituent, and (3) no substituents were 65.3, 31.5, and 3.2%, respectively. The specific rotation of galactomannans was shown to correlate with their content of galactose.

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin.

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.

Characteristics of protein-aqueous medium interactions measured by partition in aqueous Ficoll-Dextran biphasic system.

Partitioning of a number of proteins in the aqueous Ficoll-400-Dextran-70 biphasic system was studied at pH 7.4 under varied ionic compositions. The relative hydrophobicities of the proteins have been estimated, and the contributions of the interactions of the ionogenic and non-ionic groups of a protein with an aqueous environment to the total hydrophobicity of the protein have been evaluated. Some arguments in support of the biological significance of the effect of ionic composition on the relative hydrophobicity of biological macromolecules are given. Possible applications of the partition technique to protein research are discussed.

Physico-chemical factors governing partition behaviour of solutes and particles in aqueous polymeric biphasic systems. III. Features of solutes and biological particles detected by the partition technique.

The partition behaviour of human serum albumin and oxyhaemoglobin and several amino acids and small peptides was studied in the aqueous Ficoll-dextran biphasic system as a function of the ionic composition and pH. The partition coefficients of the solutes were expressed in terms of the equivalent number of CH2 groups, nCH2, and the equivalent number of carboxyl groups, m. The physical meaning of these two parameters and of the relationships found between them and pH for the proteins examined are discussed. A correlation was established between the difference in the relative hydrophobicities of the individual phases of various water-organic solvent systems and the interfacial tension, gamma 12, of the systems. It is argued that a relation of a similar type exists for the aqueous polymeric biphasic systems. The possibility of estimating the relative intensity of Van der Waals and hydration interactions of a solute and particle surface by examination of their partitioning in a biphasic system calibrated for the hydrophobic and hydration properties of the phases is discussed.