Study of snake venom derived anti-platelets aggregation / Шинэ санаа Шинэ нээлт

Snake venom toxins have been reportedly used as a rich source of a number of proteins of biotechnological interest due to their wide range of effects on haemostasis. These effects vary greatly: coagulant, anticoagulant, platelet-activating, anti-platelet, fibrinolytic and hemorrhagic, in either enzymatic or non-enzymatic pathways. Agkistrodon venom contains a variety of proteins that possess antiplatelet activities. This study presents recent development in our laboratory to produce and purify antiplatelet proteins derived from Agkistrodon blomhoffi ussuriensiss nake venom. Different matrices of HPLC (size exclusion, ion exchange and affinity chromatography) were employed for purifying the proteins and their biological and biochemical properties were characterized by SDSPAGE, 2-D electrophoresis, platelet aggregation assay and enzyme activity assay.A purified disintegrin was a single chain glycoprotein with Mr of 13 kDа and рІ 4.7, whereas PLA2 had Mr of 14 kDa and pI of 4.17. A dose-dependent activity curve analysis shows that the platelet aggregation inhibitory activity of disintegrin and PLA2 in the rabbit plateletrich plasma were ID50 of 0.25 μМ and 0.65 μМ, respectively. Bioprocesses to produce and purify active antiplatelet agents from A. blomhoffi ussuriensis venom have been developed, using modern liquid chromatography matrices. Ongoing work to optimize large-scale production process is being undertaken.

Role of A-chain in functioning of the active site of human alpha-thrombin.

This review summarizes current data suggesting that A-chain of the human alpha-thrombin molecule plays a role of allosteric effector in catalytic reactions with various substrates. Special attention is paid to the relationship between A-chain structure and catalytic activity of thrombin. The existence of this relationship is based on studies of natural mutation of A-chain of the alpha-thrombin molecule. Use of molecular and essential dynamics confirmed the role of A-chain in changes of conformation and catalytic properties of this enzyme; these changes involve residues located in the specificity sites and some inserting loops. Current knowledge on structure and properties of thrombin can be used for the development of new antithrombin agents.

[Sodium ions as the effector of catalytic action of alpha-thrombin].

A process of thrombin interaction with synthetic and natural substrates in the presence of Na+ ions has been analyzed in the survey. Molecular bases of this interaction have been presented, interrelation between the structure and function of thrombin has been noted; the nature of the unique site of its active centre which determines high thrombin affinity for the substrates and increase of its catalytic activity defined by the term of "specificity to univalent cations" have been considered in detail. Na+ ions play the role of allosteric effector in realization of two informational states of thrombin which penform, respectively, two fundamental and competing functions in the process of hemostasis. The molecular basis of the process of Na+ binding with thrombin is rather simple and depends only on the single site which importance for the enzyme function is marked by numerous investigations of a number of authors, and it is shown that Na(+)-binding site is distributed in the other zone of thrombin molecule as compared to exosites I and II, which do not take part in Na(+)-binding and allosteric transduction. Considerable attention was given to conformational conversions of a thrombin molecule caused by Na+ ions binding. It was shown that the transition slow <--> fast of the enzyme forms leads to formation of the ion pair Arg-187: Asp-222, optimal orientation of Asp-189 and Ser-195 for binding of substrates and considerable shift of the lateral chain Glu-192 determined by the disturbance of the lattice of water molecules which connects Na(+)-binding site with aminoacid Ser-195 of the active centre of the enzyme. New data have been presented which indicate that the changes in the lattice of water molecules and allosteric nucleus of Na(+)-binding site of the enzyme are the basic link of raising the affinity between the thrombin and substrate and mechanism of the enzyme activation by Na(+)-ions. The survey touches some problems of creation of allosteric inhibitors of thrombin which can take essential effect on Na(+)-binding site and favor stabilization of the anticoagulant slow-form of thrombin, and of enzyme rational mutants with selective specificity in respect of protein C which display effective and safe anticoagulant and antithrombotic effects in vivo.

[Technology of immunoglobulin production. I. Technological aspects of purification].

Plasma-derived intravenous immunoglobulin (IVIG) preparations have become the major plasma products on the world blood product market due to the successful application for the prophylactic prevention of infectious diseases and replacement therapy in autoimmune and inflammatory diseases. In this review classical manufacturing processes as well as new chromatographic, membrane, and mixed industrial technologies are discussed with respect to the cost and amount of the final product of high quality and virus- and prion-safety which is to be obtained.

Induction of catalytic activity of plasminogen by monoclonal antibody IV-Ic in the presence of divalent metal cations and alpha2-antiplasmin.

Investigation of the influence of divalent metal cations on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic showed that the presence of metal cations in the reaction medium changes the induction by slowing down or accelerating the process. Ions of Zn(2+), Mn(2+), and Cu(2+) completely inhibit activation. Ions of Co(2+) and Ni(2+) decrease the rate of the first and second phases of the reaction more than 2 times. Ca(2+) ions do not have any effect on the activation rate. Ions of Mg(2+), Ba(2+), and Sr(2+) increase the rate of the first phase of the reaction by 1.5, 2.0, and 2.0 times and the rate of the second phase by 2.0, 3.8, and 4.7 times, correspondingly. Sr(2+) ions have the strongest stimulating effect on plasminogen activation by monoclonal antibody IV-Ic. Investigation of the dose dependent effect of Sr(2+) on the rate of plasminogen activation by monoclonal antibody IV-Ic showed stimulating effect of Sr(2+) at concentrations from 0.1 to 1.0 mM with half maximum at 0.6 mM. However, Sr(2+) ions do not affect amidolytic activity of plasmin and activation of plasminogen by streptokinase. Sr(2+) ions also do not affect monoclonal antibody IV-Ic binding to plasminogen. The effect of Sr(2+) is specific and mediated by the IV-Ic component. The presence of metal cations affects conformational changes in the process of active site formation. Metal cations also affect structure of the plasminogen molecule active site in the complex with monoclonal antibody IV-Ic and enzyme-substrate interaction. The effect of alpha(2)-antiplasmin on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic in range of concentrations from 5 to 30 nM has been studied. alpha(2)-Antiplasmin at concentration 30 nM almost completely inhibits induction of plasminogen catalytic activity by monoclonal antibody IV-Ic at the ratio plasminogen/alpha(2)-antiplasmin of 3 : 1. This can be explained by competition of alpha(2)-antiplasmin and monoclonal antibody IV-Ic for the lysine-binding sites of plasminogen and inhibition of the active center in activated complex plasminogen*-mAB IV-Ic. Divalent metal cations and alpha(2)-antiplasmin are important factors in induction of plasminogen catalytic activity by monoclonal antibody IV-Ic.

[Human thrombin: drug stability and stabilization].

Stabilization of enzymes is a key factor when using biocatalysis in practice. Each enzyme stability depends both on the structure of its molecule and on the effect of various environmental factors, thus, one of the methods of the enzyme stability preservation is the formation of optimal macromedium. Thus, water structure and enzyme hydration change in the presence of solvable additives that affects its stability and catalytic properties. The paper deals with a new method of stabilization of human thrombin developed by the authors. It is proposed to use some known organic-ligands which have ion group and different nonpolar hydrophobic groups instead of traditional additives (salts, aminoacids, polyols, polyethylene glycols etc.). Thrombin stabilization proceeds in the conditions something changed compared with traditional ones. Processes of thrombin stabilization by the above compound have been investigated, enzyme stability at different temperatures and long-term storage of diluted solutions of the preparation in different conditions have been studied. It has been established that rosselin and orange II are the most efficient ligands. Optimal finite concentrations of stabilizing agents make approximately 0.0012-0.0014 M which are rather low in the system thrombin-ligand. It has been found that diluted solutions of thrombin are more stable, than concentrated ones. In the latter case the process of autolysis is included that affects negatively the catalytic effect of the enzyme, as far as there occurs the change of thrombin molecule structure, especially of thrombin beta-chain sections, evoking conformational changes of some sites of its active centre. The experiments directed to increasing thrombin intensity in the presence of organic ligands rosselin and orange II are discussed in details. Special attention is given to autolytic method of thrombin inactivation. It is admitted on the basis of already obtained data that thrombin binding with organic ligands proceeds at the expense of anionic area of beta-domain of thrombin active centre where basic aminoacids arginin and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.) were found. Under these conditions the hydrophobic interaction is provided at the expense of apolar binding of thrombin active centre area.

Immobilization of mouse single-chain antibodies for affinity chromatography using the cellulose-binding protein.

A laboratory method for obtaining immunoaffinity medium for chromatographic purification of recombinant human interferon alpha2b (IFN-alpha2b) is described. The method is based on oriented and non-covalent immobilization of recombinant antibody fragments on cellulose. The single-chain fragment variable (ScFv) against human IFN-alpha2b was genetically fused to cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome and expressed in Escherichia coli. After the isolation of the target protein in functionally active form from bacteria cells its bioaffinity immobilization on several forms of cellulose powders has been carried out. The crystalline microgranular cellulose with immobilized ScFv-CBD-fusion protein was used as affinity medium to perform the purification of recombinant human IFN-alpha2b directly from clarified extract of E. coli cells.

[Purification of biomolecules by the method of the expanded bed adsorption chromatography. III. Method optimization. Aplications].

This review introduces the principles of the expanded bed adsorption (EBA) and serves as a practical guide to the use STREAMLINE adsorbent and columns available on the market. Critical operational parameters will be discussed as well as the principles for the method design and optimization that will ensure maximum operation of this unique unit. The review is illustrated with the examples of different types of biological molecules which have been purified when using the expanded bed adsorption.

Thermodynamic characteristics of plasminogen activation by indirect activators.

Several indirect plasminogen (Pg) activators are known including streptokinase and the monoclonal antibody IV-Ic, whose mechanism of activation is well studied. To characterize thermodynamically the activation of Pg by streptokinase (SK) and the monoclonal antibody (mAB) IV-Ic, the activation energies were calculated for various reaction stages. Activation energy of 7.4 kcal/mol was determined for the interaction of the chromogenic substrate S-2251 with plasmin (Pm) and activated equimolar complexes Pm-SK and Pg*SK at the steady-state reaction stage, and 18.7 kcal/mol with the complexes Pg*IV-Ic. A 2.5-fold increase in the energy of activation for the Pg*IV-Ic complex suggests a more intricate mechanism of its interaction with the substrate. At the stage of increasing active center concentrations and the formation of activated complexes Pg*SK and Pg*mAB IV-Ic, the activation energy was found to be 10.5 and 38 kcal/mol, respectively. At this reaction stage the conformational rearrangement of Pg molecule with the formation of active center is the limiting stage determining the reaction rate. Unexpectedly high energy of activation at the second stage of interaction between mAB IV-Ic and Pg suggests several simultaneous reactions and complexity of conformation rearrangement in the Pg molecule in activated complexes, thus requiring large energy expense. Formation of the active center is probably accompanied by its transition within a narrow temperature range into another conformation state with the change in activation parameters of the reaction. Quantitative evaluation of the studied reactions from the perspective of thermodynamics of the enzymatic reactions gives more comprehensive characteristics of the activation mechanism.

[Interrelation between thrombin structure and its stability].

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.

[Purification of biomolecules by the method of adsorptional chromatography in the expanded bed. I. Principles of the method]. / Ochistka biomolekul metodom adsorbtsionnoi khromatografii v rasshirennom cloe. I. Printsipy metoda.

Principles the Expanded Bed Adsorption (EBA) have been described in the survey. The paper also deals with critical operation parameters, principles of the method design and optimization, which will guarantee maximum operation of this unique operation stage. All these problems have been discussed. The survey is illustrated by the examples of various types of biological molecules which have been purified using EBA.

[Biomoleculs purification by the method of the expanded bed adsorption chromatography. II. Design of the experiment]. / Ochistka biomolekul metodom adsorbtsionnoi khromatografii vo vzveshennom sloe. II. Proektirovanie éksperimenta.

This review introduces the principles of the Expanded Bed Adsorption (EBA) and serves as a practical guide to the use of STREAMLINE adsorbent and columns available on the market. Critical operating parameters will be discussed as well as principles for the method design and optimization which will ensure maximum exploitation of this unique operation stage. The review is illustrated with examples of different types of biological molecules which have been purified using Expanded Bed Adsorption.

[Effect of streptokinase on the interaction of alpha-2-antiplasmin with different sites of plasmin molecule]. / Vliianie streptokinazy na vzaimodeistvie alfa-2-antiplazmina s razlichnymi uchastkami molekuly plazmina.

Interaction of streptokinase and alpha-2-antiplasmin with plasmin and plasminogen fragments was compared. Binding sites on the enzyme become half-saturated, streptokinase and alpha-2-antiplasmin concentration being 8.5 and 30 nM, respectively. 6-Aminohexanoic acid in concentration of 20 mM reduces the adsorption of streptokinase and and alpha-2-antiplasmin by 20 and 60%, respectively. From all the investigated fragments, streptokinase shows the greatest affinity for mini-plasminogen and alpha-2-antiplasmin for kringles 1-3. Both proteins in the presence of 20 mM 6-aminohexanoic acid do not bind with kringle domains. Arginine dose 0.1 M does not influence streptokinase adsorption on mini-plasminogen and decreases the value of alpha-2-antiplasmin binding with mini-plasminogen by 50%. The data obtained indicate that plasminogen molecule has the sites of the highest affinity for streptokinase on the serine-proteinase domain, however for alpha-2-antiplasmin it is in the kringles 1-3. Streptokinase with equimolar quantity in respect of alpha-2-antiplasmin inhibits the adsorption of alpha-2-antiplasmin on the plasmin by 70% and in the presence of 6-aminohexanoic acid it is inhibited completely. Addition of streptokinase also increases the influence of increasing concentration of the acid. Inhibiting influence of streptokinase decreases, and that of 6-aminohexanoic acid increases, when plasmin is modified with diisopropylfluorophosphate in its active centre. At the same time maximum inhibition of streptokinase adsorption on the plasmin at different concentrations of alpha-2-antiplasmin and 6-aminohexanoic acid accounts for only 20%. We suppose that in the process of complex formation streptokinase competes with alpha-2-antiplasmin for the binding sites on the catalytic domain of the plasmin. Partial or complete blocking of the plasmin active centre contact zone by streptokinase effectively protects it from inhibition by alpha-2-antiplasmin.

[Interrelation between thrombin structure and its stability]. / Vzaimosviaz' mezhdu strukturoi trombina i ego stabil'nost'iu.

Data concerning peculiarities of fermentative nature and structure of thrombin in water-salt solution have been generalized; regularities of stabilizing effect made on thrombin by various polyols and other substances have been analyzed. It has been shown that formation of thrombin optimum macrostructure is one of the methods of its stabilization. Presence of different dissolving additives changes this enzymes hydration and this affects its stability and activity. There exist some systems to stabilize thrombin solutions. The systems consist of various salts, low-molecular and high-molecular polyols, surfactants, protein chain, composition buffer, etc. It has been shown that optimal concentrations of polyols, buffer salts and surfactants, as well as protein interaction increase considerably thrombin stability, preserving secondary structure even under its low concentration in the solution.

[Enzymes of snake venoms]. / Fermenty zmeinykh iadov.

Snakes' venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. Activators and inhibitors affecting blood coagulation and fibrinolysis systems are of special interest. Venom components can be classified into three main groups, such as procoagulants, anticoagulants and fibrinolytic enzymes according to their action. This review is focused on enzymes from Agkistrodon halys halys venom. They are thrombine-like enzyme, named Ancystron-H, flbrinogenolytic enzyme, protein C activator and platelet aggregation inhibitor. Ancystron-H is used for determination of fibrinogen level in blood plasma of patients undergoing heparin treatment and blood coagulation inhibitors accumulation. The fibrinogenolytic enzyme can be used as the instrument for protein-protein interactions in fibrinogen-fibrin system. The protein C activator is used for protein C level determination in blood plasma with different pathologies. Functions of the platelet aggregation inhibitor, belonging to disintegrins group, can be used for development of antithrombotic preparations. Information about the use of snake venoms in science and medicine is presented.

[Interaction of human alpha-thrombin with organic ligands of ionic nature]. / Vzaimodeistvie alpha-trombina cheloveka s organicheskimi ligandami ionnoi prirody.

Investigations results of human thrombin interaction with organic ligands of ion nature containing nonpolar groups are presented. It is shown that electrostatic interaction is the basic one under enzyme binding, while hydrophobic binding is only additional function in the reaction enzyme-ligand, this fact is confirmed by the absence of interaction between thrombin and rivanol which has a positive charge side by side with cumbrous hydrophobic group. New data are presented about the ligand specificity of binding sites of thrombin active centre. The importance of relative arrangement of hydrophobic ligand groups for interaction with enzyme is shown. It is supposed that thrombin binding with organic ligands occurs owing anionic site of beta-domain of active thrombin centre with the major aminoacids arginine and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.). It is shown that the compounds containing negative group SO3 and have some cunbours hydrophobic groups interact more intensively with the enzyme. Thus, rosseline--with symmetrical hydrophobic nucleus (four benzene rings)--is the most efficient ligand for the binding with thrombin. The obtained investigation results evidence for bacteriostatical and stabilizing effect of low-molecular asobenzene ligands on rather labile thrombin molecules.

[Blood protein technological industrial developments as a mirror of fundamental studies bgy the Institute of Biochemistry of the Ukrainian National Academy of Sciences]. / Tekhnolohichni rozrobky vyrobnytstva bilkiv krovi iak viddzerkalennia fundamental'nyk doslidzhen' systemy zsidannia krovi v instytuti biokhimiï nan Ukraïny.

We have examined the technology for an industrial chromatographic production highly purified factor VIII concentrate intended for therapy of the hemophilia A and characterized this factor VIII. The final product has been prepared from cryoprecipitate of pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on AEM and CEM ion exchange and SPG or SHR gel filtration chromatography. The specific activity of the product was 459 +/- 19 IU factor VIII/mg protein (n = 10), corresponding to a purification factor of about 15,000. The concentrate was free of the fibrinogen, alpha-2-macroglobulin, alpha-1-acidglycoprotein, haptoglobin. Only three contaminants could be detected: fibronectin, immunoglobulins A and G (about 0.020, 0.004 and 0.034 microgram/IU factor VIII, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Another examination was concern to the technology for an industrial chromatographic production highly purified factor IX concentrate intended for therapy of the hemophilia B and characterized this factor IX. The final product has been prepared from pooled human plasma using a large-scale procedure combining four conventional chromatographic steps based on AEM ion exchange, AFM affinity and SGS gel filtration chromatography. The specific activity of the product was 149 +/- 10 IU factor IX/mg protein (n = 10), corresponding to a purification factor of about 9000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of possible contaminants were absent in this new product. High-molecular-weight kininogen, factor VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/IU factor IX, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the high purified factor IX by Institute of Biochemistry technology tested had a lower thrombogenic power than the commercial factors IX tested. The concentrate has been subjected to a special solvent--detergent treatment for definite time and temperature during its production to virus inactivation (it will be describe in following special examination). These data demonstrate that a highly purified therapeutic clotting factor VIII and IX concentrates can be prepared from human plasma by conventional chromatographic methods developed by Institute of Biochemistry of NAS of Ukraine and Combio Ltd.

The effect of long-chain N-acylethanolamines on some membrane-associated functions of neuroblastoma C1300 N18 cells.

As reported earlier (Gulaya, N.M., Vaskovsky, V.E., Vystosky, M.V., Volkov, G.L., Govseeva, N.N. and Artemenko, I.P. (1988) Ukr. Biochim. J. 60, 58-63), N-acylphosphatidylethanolamines (NAPE) and products of their catabolism, N-acylethanolamines (NAE), are present in the lipids of neuroblastoma C1300 N18 undifferentiated cells. The present paper describes the distribution of NAE added to culture medium of differentiated cells and its effect on the fast sodium channels and some other membrane characteristics. It is shown that NAE inhibits the destroying action of veratridine on membranes.

[Distribution of ]1-14C]N-palmitoylethanolamine and its metabolites in subcellular fractions of neuroblastoma C1300 N18]. / Rozpodil [1-14C]N-pal'mitoïletanolaminu ta iogo metabolitiv u subklitynnykh fraktsiiakh neiroblastomy C1300 N18.

Distribution in dynamics of [1-14C]N-palmitoyl ethanolamine in the subcellular fractions of neuroblastoma C1300 N18 has been studied. It is shown that distribution dynamics of the label is different depending on subcellular fractions. The level of the label in cytoplasm 15 min after incubation reaches the value which remains constant till the end of the experiment. In the microsomal fraction the label is accumulated with time and becomes maximum at the end of the experiment. The highest amount of the label in the plasma membrane has been found 15 min later and then its amount falls. Parallel with this the amount of free fatty acids grows and then begins to fall simultaneously with an increase of the amount of esterified fatty acids. A considerable amount of the label of fatty acids has been determined 15 min later in the composition of di- and triglycerides of cytosol and microsomal fraction.

[Fatty acid composition of rat liver chromatin fractions under conditions of stimulation of lipid peroxidation]. / Zhirnokislotnyi sostav fraktsii khromatina pecheni krys v usloviiakh stimuliatsii perekisnogo okisleniia lipidov.

The residues of non-saturated fatty acids being the substrates of lipids peroxidation in chromatin are introduced to the composition of fractions of transcriptionally active and repressed chromatin of rat liver. The amount of non-saturated fatty acids is higher in transcriptionally active chromatin fraction as compared to their amount in the repressed fraction which correlated with the level of lipids peroxidation in these fractions under its activation in vitro. Stimulation of lipids peroxidation in vivo as a result of a single administration of tetrachloroethane and under E-avitaminosis results in the expressed shift of fatty -acid composition of the both chromatin fractions: the amount of substrate lipid peroxide oxidation increases in its repressed part and decrease in its transcriptionally active part. Administration of ionol antioxidant does not result in any noticeable normalization of the shift of fatty-acid composition of chromatin evoked by the lipids peroxidation.