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Intense using of computational methods for enzyme interaction study with other compounds of organism is demonstrated. Importance of such approach and increasing interest of researchers to glycosaminoglycan ligand reaction on pharmacologically significant biocatalysts with molecular docking methods is noted. Obtained with its help data contribute the disclosure of observe interaction mechanism, development of enzyme activity control and grounding of rational recommendations for novel therapeutic means development of high-molecular kind. The results of molecular docking of heparanase, chondroitinlyase ABC, chondroitinase B, and hyaluronidase were shown. The approach to rational design of molecules of compounds regulating enzyme activity for novel drug derivative development of directional action is representative. The perspectives of computational methods for glycosaminoglycan with enzyme complexion is discuss.Продемонстрировано интенсивное использование вычислительных методов для исследования взаимодействий ферментов с другими соеди- нениями организма. Отмечена значимость такого подхода и увеличивающийся интерес исследователей к изучению воздействия гликозамино- гликановых лигандов на фармакологически значимые биокатализаторы методами молекулярного докинга. Полученные с его помощью данные способствуют раскрытию механизмов наблюдаемых взаимодействий, развитию приемов управления активностью ферментов и обоснованию целенаправленных рекомендаций по разработке новых терапевтических средств высокомолекулярной природы. Показаны результаты выпол- нения молекулярного докинга с гепараназой, хондроитиназой ABC I, хондроитиназой В, гиалуронидазой. Представлен подход к рациональ- ному дизайну молекул соединений, регулирующих активность ферментов, для разработки новых лекарственных производных направленного действия. Обсуждены перспективы расчетных методов для исследования комплексообразования гликозаминогликанов с белками.

We have demonstrated the possibility of stimulation of the function of various types of precursor cells with hyaluronidase modified with chondroitin sulfate. Parenteral administration of modified hyaluronidase increased the number of fibroblast, granulomonocyte, and erythroid CFU in the hemopoietic tissue. The changes in the pool of mesenchymal progenitor cells were more pronounced in comparison with those induced by native enzyme.

Summary Objective : The aim of the study was to define thrombolytic efficacy of the combination of tissue plasminogen activator (t-PA) and urokinase-fibrinogen (uPA-Fbg) covalent conjugate using a simplified scheme of administration in vivo. Design : Plasminogen (Pmg) activation by t-PA and uPA-Fbg separately and combined was evaluated in vitro, both in the presence of fibrin-monomer (FM) and without it. We have conducted a comparative study of thrombolysis efficacy on the model of venous thrombosis using t-PA preparations and uPA-Fbg conjunction applying various schemes of administration. Materials : Recombinant t-PA and covalent conjugate uPA-Fbg (obtained earlier) which was exhibiting pronounced prolonged thrombolytic action were used in the study. Interventions : Thrombolytic efficacy of preparations was assayed on the model of 125 I-labelled venous thrombosis in dogs. Bolus, bolus-infusion and sequential double bolus administration of thrombolytics were used to compare their effects. Main outcome measures : Pmg activation by t-PA and uPA-Fbg preparations (in the presence of FM and without it) was monitored by hydrolysis of S-2390 chromogenic substrate in vitro. Thrombolytic efficacy of the preparations was evaluated by radioactive level and content of hemostatic proteins (fibrinogen (Fbg), Pmg and alpha-2-antiplasmin) in blood samples taken from dogs. Radioactivity increase in blood showed thrombolysis dynamics, while the change in hemostatic protein content showed disturbed action of thrombolytics on the fibrinolysis system. Results : The in vitro model system showed additivity of t-PA and uPA-Fbg action, both in the presence and without FM. Sequential double bolus administration of t-PA and (then after 15 min) uPA-Fbg conjugate into dogs with venous thrombosis revealed a fast and stable thrombolytic action of the conjuction mentioned (2.5 mg t-PA and 250 000 IU of uPA-Fbg), as well as its noticeable, but moderate (not exceeding 40% decrease from the initial level) influence on the indexes of systemic fibrinolysis. Thrombolytic action of t-PA in conjunction with uPA-Fbg is considerable and surpasses effects of the same doses of its components administered separately. It was significantly similar to the conjunction action after the bolus-infusion-bolus scheme of administration and the action after bolus-infusion administration of 10 mg t-PA (at later stages of monitoring). Data demonstrated eligibility of t-PA and uPA-Fbg thrombolytic composition for effective research application with a simplified scheme of administration. Conclusion : Conjunctive application of t-PA and uPA-Fbg conjugate exhibiting complementary action and various pharmacokinetic profiles is promising for efficient and safe thrombolysis with a simplified scheme of the preparation administration. Thrombolytic compositions (mixtures) on the basis of these, or similar short- and long-term acting plasminogen activators, are likely to become one of the main and widespread means of treatment of thromboembolism disease in the 21st century.

The aim of this work was to create sedimentological model suitable for field development. Novoportovskoe field was discovered in 1964, but its pilot drilling began only this year. All preserved data was compiled to understand its depositional history which is considered as the most complex in region. Seismic data didn’t provide good base for facies modeling, so mostly core and well-log data was analyzed. Lower Jurassic strata were deposited in deltaic conditions with periodical sea-level change. Middle Jurassic strata were deposited in continental conditions as fluvial sediments of channels, crevasse splays and overbank muds. The distribution of reservoirs and trends will be used for static models to maintain drilling process

A water-soluble enzymatic preparation was obtained by coimmobilization of papaya proteinase complex together with cysteine on aldehyde dextran. The product formed possesses an extended pH optimum of catalytic activity, and increased stability, including storage stability. The formation of a protective microenvironment from cysteine sulfhydryl groups favors the fixation of catalytically active enzyme conformers upon their immobilization on the polymeric carrier. The stability of native and aldehyde dextran-cysteine-modified papaya proteinase complex towards the action of extreme pH, proteolysis, and temperature was comparatively studied. It was demonstrated that the stabilizing effect of the polymeric carrier is determined by the “strengthening” of the biocatalyst structure and by the prevention of protein association via thioldisulfide exchange reaction. The latter is the results of steric hindrance created by the carrier. At extreme pH values a destruction of the preparations occurs, which increases in the presence of pronase. The partial destruction of the carrier in the modified preparation does not decrease thermostability of the latter. The modification of the preparation increases its stability and diminishes degradation at extreme pH values. The therapeutic efficiency of the modified preparation has been demonstrated at ophthalmologic lesions.

The effect of intramolecular cross-linkages of different length on the thermostability of α-chymotrypsin has been studied. To this end, the carboxy groups of the enzyme were first activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; then the activated proteins was treated by diamines of NH2(CH2)n NH2 type with n ranging from 0 to 12. The dependence of the rate constant of monomolecular thermoinactivation of the enzyme on the length of the cross-linking agent gives a minimum corresponding to tetramethylenediamine. The number of cross-linkages may be increased by enriching the protein molecule with carboxy groups; for this purpose α-chymotrypsin was succinylated. For succinylated α-chymotrypsin, the dependence of the rate constant of monomolecular thermoinactivation of the enzyme on the length of the cross-linking agent has a minimum for a shorter bifunctional reagent, ethylenediamine. In addition, the maximum stabilizing effect (compared to the native enzyme) increases (from 3- to 21-fold) of instead of α-chymotrypsin modified with tetramethylenediamine, succinylated α-chymotrypsin modified with ethylenediamine is used. This approach may be generally employed for preparation of stabilized water-soluble enzymes.

The dependence of α-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermo-inactivation (50°C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4. NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.

The denaturing action of guanidine · HCl on modified α-chymotrypsin (EC 3.4.21.1) preparations has been studied. The consecutive treatment of α-chymotrypsin with N- acetyl-homocysteine thiolactone, 5,5′-dithio-bis-(2-nitrobenzoic acid) and dithiols of HS-( CH 2 ) n -SH type, with n ranging from 4 to 10, leads to enzyme stabilization as a result of protein modification. A greater stabilization effect can be achieved by enriching the protein molecule with groups reactive towards dithiols, after first modifying carboxygroups. In this case dithiol with n = 5 forms an intramolecular cross-linkage. If an equimolecular mixture of different dithiols is used for enzyme modification, the enzyme gradually ‘selects’ 1,5-dithiol for the formation of an intramolecular cross-linkage instead of the initial one-point modification. The use of potentially reversible cross-linkages may be generally employed for the preparation of stabilized water-soluble enzymes via the mechanism of selfstabilization.