Study of the structure of streptokinase conjugates with a hydrophilic vinylpyrrolidone copolymer.

The modification of streptokinase by a synthetic N-vinylpyrrolidone copolymer leads to formation of conjugates varying in structures according to the proportions of the components in the reaction medium. Based on data obtained from spectrophotometry, as well as sedimentation and diffusion analyses, it is shown that in the presence of excess protein in the reaction medium, formation of the main chain takes place via the copolymer associated with several protein globules. Under conditions of excess modifier copolymer, either single-site and/or multiple-site bonding is possible for the protein backbone, depending on the molecular weight of the copolymer. One of the models for the conjugates obtained in this manner has been corroborated by small-angle X-ray scattering data. CD spectral analyses has been performed in order to demonstrate that covalent modification does not alter the secondary structure of streptokinase in the conjugate whereas the tertiary structure undergoes local changes in conformation.

[A study of native streptokinase and its polymeric derivative by means of small angle x-ray scattering]. / Issledovanie nativnoi streptokinazy i ee polimernogo proizvodnogo metodom rentgenovskogo rasseianiia pod malymi uglami.

The gyration radius (R0) of native streptokinase (SK) was found to be R0 = (40 +/- ) A by small-angle X-ray scattering. Experimental hydrodynamic characteristics of SK were S0(20),W = (2.8 +/- 0.1)S; D0(20),W = (6.0 +/- 0.5) x 10(-7) cm2/s; [n] = 0.12 dl/g. The molecular weight of the enzyme was found to be 44,000. The values of the form factor R0/Rsphere = 2.1 and the frictional ratio f/f0 = 1.5 indicate considerable anisometry of the SK molecule. Basing on the curves of small-angle X-ray scattering of SK modified with a synthetic linear copolymer of N-vinylpyrrolidone (P) at a molar ratio SK less than P, a structural model of the conjugate was proposed. The modified form consisted of a dense nucleus covered with a diffuse polymeric membrane. In accordance with the model, R0 of modified SK and of the whole conjugate were found to be R0nucleus = (34 +/- 2) A and R0conjugate = (114 +/- 5)A.

[Chemical modification of proteolytic enzymes by low molecular weight agents]. / Izuchenie khimicheskoi modifikatsii proteoliticheskikh fermentov nizkomolekuliarnymi agentami.

Low-molecular modification of proteolytic enzymes with aldehydes and anhydrides of carboxylic acids as well as with 2,4,6-trinitrobenzene sulphonic acid was studied. Specific activities of the enzymes were found to be dependent on the modification degree of their amino groups. The retaining of high activities in the region of low extents of enzyme modification enabled biocatalysts with activities similar to those of the native enzymes to be prepared.

[The conformation properties and conformation stability of streptokinase and its polymer derivative]. / Izuchenie konformatsionnykh svoistv i konformatsionnoi stabil'nosti streptokinazy i ee polimernogo proizvodnogo.

The properties and conformational stability of the proteinaceous activator of fibrinolysis--native streptokinase--and its derivative obtained by modification with a linear hydrophilic copolymer based on N-vinylpyrrolidone, were studied by the circular dichroism method. It was shown that polymeric modification of streptokinase had no effect on the secondary structure, while the conformational stability of the modified protein to urea was higher than that of the native one. Studies on thermal stability of both native and modified forms of streptokinase showed that the inactivation rate was lower in the modified form as compared to the native one.

[Ion-exchange chromatography on cholesterol oxidase from Actinomyces lavendulae on a Biocarb-type carboxyl cationite]. / Ionoobmennaia khromatografiia kholesterinoksidazy iz Actinomyces lavendulae na karboksil'nom kationite tipa Biokarb.

Reversible sorption on the Biocarb-D carboxyl cationite of cholesterol oxidase extracted from the mycelium of Actinomyces lavendulae was examined. Sorption at pH 5.0 and desorption at pH 5.5 maintained 80% of the enzyme activity and increased seven-fold the specific activity as calculated per protein. By gel chromatography on Sephadex G-150 it was shown that the process was accompanied by a significant decrease in the number of inactive proteins.

[Mechanism of thermal denaturation of native trypsin and trypsin modified by neutral soluble polymer]. / Mekhanizm termodenaturatsii tripsina nativnogo i modifitsirovannogo neitral'nym rastvorimym polimerom.

The thermal denaturation of trypsin solutions and trypsin vinylpyrrolidone-acrolein copolymer conjugate was studied within the temperature range of 37.5-96 degree. pH 4.5. A mechanism for trypsin thermal denaturation based on its ability to form dimers and determining the minimal stability of the enzyme at 66 degree and its maximal stability at 74 degree is proposed. The thermal denaturation of the trypsin conjugate is of a different type, which depends on the changes in the thermal denaturation mechanism due to its incapability to form dimers. A decrease in the activation and thermodynamic parameters of the enzyme denaturation during its modification is due to the approximation of the protein structure to that of the denatured form, eventually resulting in the enzyme destabilization. The increased stability of the modified protein to the denaturating effects of low temperatures is due to the changes in the denaturation mechanism induced by enzyme modification.

[Effect of polymeric modification on specific properties of soya bean inhibitor]. / O vliianii polimernoi modifikatsii na spetsificheskie svoistva ingibitora iz soevykh bobov.

Chemical modification of the trypsin inhibitor from soya beans by a hydrophilic synthetic copolymer of N-vinylpyrrolidone was studied. The optimal conditions for modification (pH, ratio of reactants, reaction time) were established. The soluble polymeric forms of the inhibitor differing in the number of covalent bonds with the copolymer were obtained. An affinity adsorbent for isolation of trypsin was prepared by adsorption of the polymeric form on silochrome.

[Effect of chemical modification on proteinase interaction with inhibitors, on their coagulability and acute toxicity]. / Vliianie khimicheskoi modifikatsii na vzaimodeistvie proteinaz s ingibitorami, na ikh koaguliruiushchuiu aktivnost' i ostruiu toksichnost'.

A comparative study of terrylytin, trypsin and products of their covalent binding to human serum albumin was carried out. Modification caused a decrease of the affinity of both enzymes for proteinase inhibitors from the blood. The inhibition constant for terrylytin was increased 3-4-fold, that for trypsin - by 2 or 3 orders. The recalcification time of human blood plasma in the presence of terrylytin remained practically unchanged after enzyme chemical modification. Trypsin binding to albumin decreased its coagulability. Preparations of modified enzymes were characterized by a decreased acute toxicity, this effect being especially well-pronounced in the case of trypsin. The decrease of terrylytin toxicity due to modification can be accounted for by a decrease in its affinity for alpha2-macroglobulin. The decrease of trypsin toxicity is apparently due to a decrease of its coagulability due to binding to albumin.

[Physico-chemical properties of terrylytin modified by a copolymer based on vinylpyrrolidone]. / Izuchenie fiziko-khimicheskikh svoistv proteoliticheskogo fermanta terrilitina, modifitsirovannogo sopolimerom na osnove vinilpirrolidona.

The proteolytic activity of terrylytin produced by the culture of Asp. terricola and modified by a water-soluble copolymer of vinylpyrrolidone and acrolein remained unchanged after enzyme modification. Using micro-thin layer chromatography, it was shown that the bulk of the epsilon-amino groups of lysine residues of the protein enter the reaction with the aldehyde groups of the polymeric matrix. The sedimentation and diffusion patterns of the polymerenzyme adduct demonstrated that the molecular weight of the modified enzyme is the total of molecular weights of its constituent components. Evidence from viscosimetry and gel chromatography allowed to develop a hydrodynamic model of the macromolecular product. It was shown that the rate of the enzyme inactivation in the solution calculated from the first order reaction equation depends on the nature of the enzyme electrochemical microenvironment. Under conditions close to physiological ones the rate inactivation constant for terrylytin modified by a neutral polymeric matrix is 10 times less than that for the native enzyme. At the isoelectric point (pH 4,6) a positively charged polymeric form of terrylytin is found to be the most stable one. The pH and temperature optima for casein hydrolysis remained unchanged throughout polymeric modification. The polymeric membrane did not hamper the diffusion during approximation of the substrates (casein and insulin) to the enzyme molecule during the catalytic act, which manifested itself in a constancy of Michaelis curves. Terrylytin modification by a copolymer causes an increase of stability with respect to trypsin proteolysis and a decrease of human blood plasma affinity for the inhibitors. The apparent inhibition constants for modified enzyme forms do not depend on the nature of electrochemical microenvironment and exceed that for native terrylytin 10-fold.

[Kinetics of low molecular weight substrate hydrolysis by immobilized trypsin]. / Issledovanie kineticheskikh zakonomernostei gidroliza nizkomolekuliarnogo substrata immobilizovannym tripsinon.

The catalytic properties of trypsin immoblized on silochrome were studied in a flow reactor with replacement. The hydrolysis of methyl ester N-n-tosyl-L-arginine obeys the Michaelis--Menten kinetics. The apparent K'm value for the system with immobilized trypsin is considerably lower than for native trypsin. The K'm value was decreased with an increase in the rate of the substrate flow through the reactor or when smaller-sized silochrome granules were used. It is assumed that the apparent K'm value for the immobilized system is due to diffusion. The effects of diffusion on the catalytic properties of the immobilized enzyme were estimated.

[Some peculiarities of trypsin and heparin interaction]. / Nekotorye osobennosti vzaimodeistviia tripsina s geparinom.

The interaction of trypsin with an acid polysaccharide, heparin, at pH 4.2 and 8.0 is studied. Heparin is found to destabilize the enzyme under condition of both autolytic denaturation (pH 8.0) and thermoinactivation (pH 4.2). Data on trypsin inactivation kinetics suggest that the stage of forming molecular complexes with different contents of trypsin and heparin precedes the stage of the enzyme denaturation. Maximal trypsin inactivation rate takes place under equimolar enzyme:heparin ration.

[Trypsin immobilization on a mineral matrix]. / Immobilizatsiia tripsina na mineral'noi matritse.

Trypsin immobilization on the mineral matrix--silochrome was studied. The effect of the matrix electrochemical nature on the process was examined. pH-optima for trypsin binding with different silochromes and pH-optimum of action of the immobilized enzyme on casein were determined. The effect on the trypsin-silochrome binding of different supplements--inhibitors (benzamidine), stabilizers (Ca2+) and substrates (casein, benzoyl argininamide hydrochloride) was demonstrated.

[Physico-chemical and enzymatic properties of polymeric trypsin derivatives based on dextran]. / Izuchenie nekotorykh fiziko-kimicheskik i énzimaticheskikh svoistv polimernykh proizvodnykh tripina na osnove dekstrana.

The molecular weight distribution, thermal stability during autolysis, resistance to human serum inhibitors as well as temperature optimum of native and dextran-modified trypsin were investigated. The seeming constants of autolytic inactivation and inhibition of native and modified trypsin were calculated. Trypsin polymer derivatives had higher molecular weight than the native enzyme. They also showed higher resistance to autolysis and serum inhibitors. Possible causes of the above effects are discussed.

[Denaturation of the alpha-amylase of Bacillus subtilis in an acid medium]. / Denaturatsiia alpha-amilazy Bacillus subtilis v kisloi srede.

By fractionation of purine compounds (sorbtion on the cation exchange resin KU-2 (H+), extraction, precipitation of purine compounds as Ag-complexes) a "purine fraction" of the culture liquid epiphytic bacteria No. 703 isolated from barley overground organs was obtained. The presence of isopentenyl cytokinins was demonstrated by quality reactions of the purine fraction with aromatic amines and phenols as well as by the values of Rf and UV spectrum of individual compounds examined by paper chromatography of the purine fraction.

[Some physicochemical properties of modified trypsin]. / Nekotorye fiziko-khimicheskie svoistva modifitsirovannogo tripsina

Physico-chemical properties of trypsin covalently bound with human serum albumin by glutaric aldehyde have been studied. The modification of the enzyme practically caused no changes in the pH optimum of trypsin. The inhibition of modified trypsin by inhibitors from soy beans and human blood serum has been also studied. The apparent inhibition constants have been calculated. The modification has been shown to result in a deceleration of autolytic degradation. The autolysis rate constants have been calculated at 50 degrees C.