[Thrombin and anticoagulant therapy].

New data which reveal rational approaches for pharmacologic control of blood coagulation process and confirm the key role of thrombin in haemostasis processes compared with other proteinases are presented in the review. Modulation of thrombin properties described in the review gives a new possibility for creating anti-thrombin preparations. Thrombin allosteria can serve a basis for development of new therapy. Creation of catalytically inert thrombin in slow-form within efficient anti-coagulant activity in vivo will allow using it in medicine as a unique anti-thrombin agent.

[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.

[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.

[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.

[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.

[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.

[Human thrombin: enzymatic properties, stability and standardization of preparation]. / Trombin cheloveka: fermentativnye svoistva, stabil'nost' i standartizatsiia preparata.

The work deals with estimation of thrombin preparation having such features as: sedimentation activity 3000-3200 NIH un. per 1 mg of protein and 97% of active centres. The enzyme isolated has been estimated according to the amidolytic activity on synthetic substrates S-2160 and BAPNA being equal 5200 and 185 milli un/mg of protein, respectively. According to the electrophoresis in PAAG in the presence of Ds-Na the preparation is homogenous, its molecular mass is 36000. The fibrinogen sedimentation time dependence on the isolated thrombin concentration has been estimated as well as the comparative analysis with the thrombin of the firm "Sigma" with the previously calibrated activity using the international standartion (coded P4) has been conducted. The absence of proportionality between the substrate sedimentation time and the preparation concentration has been determined. It has been revealed, that if the experimental findings are presented in the units 1/t against the thrombin units NIH the right lines are received within the limits used. The defreezing and secondary freezing of the preparation preserved under -20 degrees C have been showed as rendering an essential effect on thrombin activity. In order of the enzyme stabilizing at preserving the thrombin isolated has been concentrated applying the amycon membranes (MWCo: 30,000). While applying the thrombin water-saline solution in the conditions selected the preparation has showed itself practically stable during a year without utilizing any admixtures. The essential effect on thrombin has been found from the side of 1% glycin, 0.5% PEG, 1% saccharose and so on. The thrombin isolated high functional homogeneity, its stability permit to recommend the preparation as an operative standard.

[Binding of Kunitz-Northrop inhibitor on some sorbents]. / Sviazyvanie ingibitora Kunittsa-Nortropa na nekotorykh sorbentakh.

This work is devoted to the problem of sorption and desorption of Kunitz--Northrop inhibitor on different sorbents. By passing through Dowex 1.10 column 0.05 M glycine-NaOH buffer, pH 10, two fractions with 100% inhibitor activity were identified, while different admixtures and inert proteins remained resin-bound. In trypsin-Spheron 300, trypsin-agarose and anhydrochymotrypsin-Spheron columns the contamination of sorbents-bound inhibitor complex was eliminated by washing out with 0.1 M NaCl, pH 8.0. The resin-bound inhibitor was released at pH 1. The specific activity of the preparation obtained was shown to increase in 200-240-fold, but in the case of anhydrochymotrypsin-Spheron 300 the inhibitors activity was detected both at pH 8.0 and pH 1.7. In this case the increase in specific activity was only 2 and 68-fold, respectively. The most effective inhibitor-peptide desalting was defined at application of dialysis membranes "Spectra/Por" (MWCo: 3000-5000 USA). While applying PAAG electrophoresis the standard SDS-system were shown to be ineffective. Therefore some modifications of this method were used. Being compared with molecular weight of Contrycal and other known peptides this preparation revealed the presence of protein contamination. Within 1-9 mg the peptides demonstrated a linear dependence in trypsin inhibition. The weight and molar ration of inhibitor: trypsin was found to be 1:1 and 3:1, respectively. It was calculated that IUE of the inhibitor had inhibited 0.73 mg of trypsin, IUE of Contrycal--0.15 of the enzyme, that was 4.6 fold less effective than the separated peptide.

[Polypeptide serine proteinase inhibitors isolated from venom from several reptilian species]. / Polipeptidnye ingibitory serinovykh proteinaz, vydelennye iz iadov nekotorykh vidov reptilii.

The survey encompasses literature data on the polypeptide inhibitors of some reptiles serine proteinases and their separation from adder Viperidae and cobra Elapidae species. The evolutionary comparison of physico-chemical and biological properties of them are also given and discussed within this work. Considerable homology (about 50%) in amino acid composition of adder, bee, mammal and others of different phylogenetic origin is being emphasized and high homology in structure of their functionally important inhibitors sites is observed. In the most cases the investigated peptide inhibitors of adder and cobra were observed to have an extremely high antitryptic activity with Ki ranging from 7.6 x 10(-10) M to 3.5 x 10(-12) M. The majority of polypeptide inhibitors are suggested by Laskowsky et al to interact with the proteinases in a standard way. The biological reactivity of the above preparations is a result of arginine and lysine presence in the substrate-binding sites of P2' and P3' or P4' centres.

[Isolation and properties of fish chymopsin]. / Vydelenie i nekotorye svoistva khimopsina ryb.

A simple and convenient method of fish chymopsin isolation from the acetone powder of pyloric appendices has ben developed using classical methods. The enzyme preparation includes 62-65% of protein, the specific weight of trypsin and chymopsin being 1000 and 3000 units per 1 mg of protein, respectively. Heterogeneity of the isolated chymopsin was shown using electrophoresis in PAAG. As to the degree of purity the given preparation is like to the bull chymopsin. Under identification of amino acid composition it is shown that the both chymopsins differ from each other to a certain extent. The preparation isolated from fish includes the less content of lysine that probably can evidence for the overwhelming quantity of serine proteases in anionic form as well as the less content of serine and leucine. At the same time the higher content of threonine, glutamic acid, proline, methionine is revealed in the fish chymopsin. Enzymatic properties of fish chymopsin as to the digestion of some low-molecular substrates have been studied. A comparative characteristic of their hydrolysis by the isolated preparation, bull chymopsin and pylochymopsin (fish preparation isolated on the biospecific sorbent) is presented. At any case the degree of hydrolysis of substrates by the both chymopsines is similar and to some extent different from the catalytic action of pylochymopsin.

[Peptide hydrolases of marine organisms]. / Peptidgidrolazy morskikh organizmov.

The survey is devoted to the description of properties of proteolytic enzymes of some sea organisms. Structure peculiarities and properties of proteinases of trypsin and chymotrypsin type, carboxypeptidases A and B, aminopeptidase and leucine aminopeptidase of molluscs, stars, shrimps, fishes and other sea organisms have been considered. Data are presented about trypsins typical of the sea organisms which are characterized by high content of asparaginic and glutaminic acids and small values of activation energy of the reactions which they catalyse. Data are discussed concerning stability of enzymes as to heat denaturation, effect of the environment with external values of pH. Based on the similarity of the substrate specificity of enzymes, their sensitivity to inhibitors, it is concluded that the enzymes of the sea organisms and mammals are similar.

[Role of S'2-stimulation of serine proteases in regulation of proteolysis]. / O roli S'2-stimululiatsii serinovykh proteaz v reguliatsii proteoliza.

Data about ligand specificity and functionality of the nearest surrounding of hydrolytic centre of serine proteases are presented. A regulatory role of S'2-site predetermining highly-specific activation of proenzymes into the enzymes and formation of stable enzyme-serine complexes is discussed.

[Hydrophobic interactions of serine proteases with low molecular weight compounds: partial effector displacement from the activated enzyme by the substrate]. / Gidrofobnye vzaimodeistviia serinovykh proteaz s nizkomolekuliarnymi soedineniiami: chastichnoe vytesnenie substratom éffektora iz aktivirovannogo fermenta.

1,2,3-tris-tri-beta-phenylethylaminopropane (a compound which can simultaneously interact with S1- and S2'-enzyme sites and has complicated grouping in the region analogous to "leaving group" of a substrate) has been studied for the effect on chymotriptic hydrolysis. Anomalously high inhibitory effect of some cumbersome organic compounds is explained on the base of the obtained data.

[Hydrophobic interactions of serine proteases with low molecular compounds: role of the S'2-site in substrate activation and interaction with serpines]. / Gidrofobnye vzaimodeistviia serinovykh proteaz s nizkomolekuliarnymi soedineniiami: rol' S'2-uchastka v substratnoi aktivatsii i vzaimodeistvii s serpinami.

An attempt is made to simulate the P1-P'2 site of the reactive centre of protein inhibitors of serine proteases (serpines). On the basis of data from literature structure requirements are formulated and compound 1,5 bis-dibenzyl-aminopentane is synthesized. It may simultaneously interact with S1- and S'2-sites of chymotrypsin and contains no bonds adequate to the hydrolytic centre of proteinase. The compound is studied for its effect on hydrolysis of low-molecular substrates and proteins by chymotrypsin. Results obtained are discussed as well as the possible role of the S'2-binding site in the substrate activation of serine proteinases and their interaction with serpines.

[Enzyme activity and lipids of a preparation from the pyloric ceca of salmon]. / Fermentativnaia aktivnost' i lipidy preparata piloricheskikh pridatkov lososevykh ryb.

Proteolytic activity and lipid composition of enzymic preparations of water extract from the pyloric caeca of salmon fishes have been studied. Phospholipids and sterols are tightly bound with proteins. The participation of lipids in the proteolytic activity is discussed.

[Comparative study of the properties of serine proteases of lower and higher vertebrates]. / Sravnitel'noe issledovanie svoistv serinovykh proteaz nizshikh i vysshikh pozvonochnykh.

Results of the comparative study of trypsin- and chymotrypsin-like serine proteases from pyloric caeca of salmon fishes and trypsin and chymotrypsin of bulls are presented in the paper. The hydrolytic activity of salmon proteases with respect to methyl ethers of N-benzoyl-L-leucine is 2.4 times higher than that of bull chymotrypsin, but with respect to methyl esters of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine the activity of salmon proteases is 6.5 and 80 times lower than that of bull chymotrypsin and trypsin. Salmon proteases in contrast to bull trypsin and chymotrypsin hydrolyze but slightly N-glutaryl-L-phenylalanine para-nitroanilide. It shown that fish proteases are not absolutely specific to synthetic substrates, which is a result of their less pronounced (than in case of bull trypsin and chymotrypsin) differences in structures of binding centres. The study of the salmon protease interaction with some immobilized ligands has confirmed the higher affinity of enzymes to reagents with two space-separated aromatic rings in their composition. It is supposed that salmon proteases interact with such reagents through two sites: hydrophobic "pockets" and probably additional binding site of the active centre. The salmon protease preparation demonstrates higher resistance to inactivating action of formaldehyde within the range of concentrations 2-16% than bull chymotrypsin does.

[Hydrophobic interaction of alpha-chymotrypsin with low molecular weight compounds]. / Gidrofobnye vzaimodeistviia alpha-khimotripsina s nizkomolekuliarnymi soedineniiami.

Data on alpha-chymotrypsin interactions with hydrophobic low-molecular compounds have been generalized. Existence of two sites of noncovalent interaction with hydrophobic nuclei of a ligand molecule is shown. When the substance to be bound contains only one hydrophobic nucleus, the interaction is mediated by a "hydrophobic pocket" of the enzyme--a binding site of amino acid residues which are, in the P1-position relative to the cleaved bond. Under these conditions substances with an asymmetric hydrophobic nucleus (of the tryptophan type) are better ligands for binding. In case of compounds containing several hydrophobic groups scattered in the space, interaction with the enzyme proceeds in two binding sites. New data are presented on the ligand specificity for binding sites of chymotrypsin in lower vertebrates. Relative position of hydrophobic groups of the ligand is shown as that of great importance for interaction with the enzyme. It is concluded that the binding sites of trypsin- and chymotrypsin-like proteinases of the lower vertebrates differ but less from each other as compared to binding sites of trypsin and chymotrypsin in mammals.

[Preparation and properties of trypsin from the pyloric caeca of Pacific Ocean salmon]. / Poluchenie i svoistva tripsina iz pilorichekikh pridatkov tikhookeanskikh lososei.

Trypsin from pyloric caeca of Pacific salmon was purified by affinity chromatography of the water extract on hexamethylenediamine-glycidylmethacrylate-cellulose. A protein band with a molecular weight of 22.5 kDa was found on SDS-electrophoresis in PAG. The protein band was homogeneous according to isoelectrofocusing in PAG (pI 4.0). The amino acid composition of the enzyme is typical of trypsin anionic forms; the major difference from the cationic forms is the lower content of lysine. The differences in properties caused by change of the enzyme molecule charge are similar to those observed in cationic trypsin when the lysine epsilon-amino groups of the latter are modified (change of pI, shift of the pH-optimum towards basic values, increase of stability to autolysis). Some natural trypsin inhibitors of the different origin suppressed the enzyme activity of trypsin from Pacific salmon in typical stoichiometric ratios. An unusual interaction of the enzyme with the specific inhibitor N-L-tosyl-L-lysine chloromethyl ketone was observed.

[Trypsin-, chymotrypsin-like proteinases in fishes]. / Tripsin-, khimotripsinpodobnye proteinazy ryb.

Recent data on the nature of trypsin-, chymotrypsin-like proteinases of fish are generalized. Localization and secretion of these enzymes in pyloric appendages of fish are considered in detail. Trypsin and chymotrypsin are in the state of proenzymes and transform into the active form by means of their own proteolytic factors. It is observed that the classical methods for isolation of individual chymotrypsin and trypsin cannot be used in the case of fish, since the fish enzymes are stable in the neutral and low-alkaline media and unstable in the acid medium. This is, first of all, accounted for by differences in the physicochemical characteristics of the test enzymes. New data on the biospecific chromatography of serine proteinases of lower invertebrates are presented. Biospecific sorbents used for isolating enzymes from mammals are not always convenient for purification of fish serine proteinases. This evidences for considerable differences in their active sites and, probably, in their binding sites, whose nature is responsible for the specificity and is important for the selective chromatography of enzymes.