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Different components of the renin-angiotensin system (RAS), whose content and activity are predetermined by local factors, are generated in the ocular tissue structures. The local eye RAS plays an important role in pathogenesis of different eye diseases and in the local manifestations of general pathological processes. Therefore, a study of the eye RAS components in norm and in disease contributes to understanding the pathogenesis of eye diseases and opens up new possibilities for an adequate treatment. The RAS components in the lacrimal fluid can be an important characteristics of such diseases like keratitis, diabetic retinopathy etc.

The kinetic behavior of proteolytic enzymes immobilized in thermosensitive hydrogels was studied at the phase transition (collapse) of the carriers. The dependence of the activity of immobilized enzymes on the state of the matrix allows the use of the hydrogel phase transition to regulate the activity of immobilized enzymes. Several cases of such regulation were demonstrated.

The structure of the conjugate of Bowman-Birk soybean proteinase inhibitor (BBI) with the block copolymer of ethylene oxide and propylene oxide (proxanol) containing five moles of proxanol per mole of protein, has been studied. Data from reverse phase hydrophobic HPLC suggest that the conjugate is less hydrophobic compared to native BBI. A shift of the second derivative UV absorption spectrum for the conjugate towards the shortwave region indicates a greater accessibility of the Tyr-59 residue localized in the interdomain region of the BBI molecule for the solvent. It has been assumed that the conjugate-induced increase in Ki for chymotrypsin may be due to both disturbances in the intact structure of the interdomain region of BBI and screening of the anti-chymotrypsin reactive center as a result of hydrophobic interactions of propylene oxide blocks of proxanol with exposed hydrophobic groups around the reactive center. Supporting evidence in favour of BBI molecule hydrophilization as a result of modification by proxanol can be derived from decreased conjugate penetration into intestinal epithelial cells as well as from the slow elimination of the conjugate from mouse blood stream.

A new method for determining the solvent accessibility of tyrosine and tryptophan residues in proteins from the wavelengths of absorption peak maxima in secondary derivatives of UV absorption spectra has been developed. Analytical expressions for calculating the number of groups in latent and exposed states of amino acid residues in protein globules were obtained on the basis of the previously elaborated mathematical model of spectral changes observed during the transition between those respective states. The new procedure was used to analyze sixteen proteins and showed a good correlation between the experimental results and literary data.

Cathepsin G stimulates the hydrolysis of elastin from bovine neck ligament catalyzed by human leukocyte elastase. Stimulation factor depends on the ratio of the enzyme concentrations and ionic strength and equals 1.0-2.0. The classical Bowman-Birk inhibitor from soya retards strongly the hydrolysis of elastin catalyzed by leukocyte elastase, cathepsin G and the mixture of both. The inhibitory effect is practically unaffected by the adsorption of the enzymes on elastin, prolongation of the enzymatic reaction and ionic strength.

Functions of proteinase inhibitors, connected with their ability to normalise proteolysis, are reviewed briefly. Arguments are suggested on favour of using the inhibitors as drugs for treatment of some diseases.

Isolation of the proteinases inhibitors, available for medicinal purposes, was described, where the inhibitor of the Kunitz type was obtained from bovine pancreas and the inhibitor of the Bowman-Birk type from soybeans. Screening of the immobilization procedures was carried out, which enabled the authors to produce the polymeric conjugates of the proteinase inhibitors exhibiting the maximal rate of activity against pancreatic proteinases and granulocyte elastases. Pharmacokinetics of the proteinase inhibitors obtained was studied. High molecular derivatives of the inhibitors from the bovine pancreas circulated in rat blood in larger quantities and longer, their total clearance was 5 times than native inhibitor preparations. The preparations containing these inhibitors from bovine pancreas exhibited a high therapeutic efficiency in treatment of rats with hemorrhagic pancreatitis and acute liver failure in rabbits.

A kinetic study of the interaction of the classical Bowman-Birk type soybean inhibitor (BBI 2-IV) with human granulocyte alpha-chymotrypsin and cathepsin G has been carried out. The K(a) values for the inhibitor-proteinase systems--alpha-chymotrypsin and cathepsin G (2.0 x 10(5) and 6.4 x 10(6) M-1 s-1, respectively) have been established.

Catalytic activity of alpha-chymotrypsin preparations covalently included in the matrix of the poly-N-isopropylacrylamide gel does not follow Arrhenius equation above the low critical temperature of the polymer dissolution. Starting from this temperature, at which the changes of polymer structure takes place (hydrophobization), the temperature increase results in a rate lowering for the chemical reaction catalyzed by the enzyme. This phenomenon is reversible. A correlation between temperature dependence of the immobilized alpha-chymotrypsin activity and the dehydration degree of the carrier is observed. The decrease of the water content in the matrix causes a change of the substrate specificity of the immobilized alpha-chymotrypsin.

A quantitative method is suggested for estimating the structure and conformational stability of proteins based on the individual absorbance of Tyr residues in the second derivative UV spectra. Subtilisins Carlsberg, BPN' and 72 were chosen as the model proteins. The values of the increase of the Tyr absorption at 282.3 nm upon the total denaturation of the proteins made it possible to calculate the number of the exposed and "buried" tyrosine residues in the native proteins. A mathematical model of spectrum changes during the transition of Tyr residues from the "buried" to exposed form is suggested. The method is useful for the determination of the denaturation constants of proteins bearing "buried" tyrosine residues.

A one-step procedure for human leukocyte elastase purification using an affinity adsorbent based on protein soybean Bowman-Birk proteinase inhibitor has been developed. The leukocyte elastase was purified 70-fold with a 70-100% yield. The enzyme preparations did not contain cathepsin G and displayed a high specific activity. The soybean Bowman-Birk type inhibitor effectively inhibited the elastin hydrolysis by leukocyte elastase both when the enzyme and the inhibitor were simultaneously added to the substrate and after preliminary elastase adsorption on elastin. The inhibitory effect was preserved at high degrees of elastin hydrolysis.

A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.

A classical soybean inhibitor (Bowman-Birk inhibitor, BBI 2-IV) and two high molecular weight glycine-enriched inhibitors of the same type (3-II and 4-II) have been isolated, purified to homogeneity and characterized. All of the BBI isoforms have been found to effectively inhibit cathepsin G and human granulocyte elastase. The constants for leucocyte cathepsin G inhibition by classical BBI 2-IV (Ki = 1.2 x 10(-9) M) and high molecular mass BBI 3-II (Ki = 8.0 x 10(-8) M) as well as for leucocyte elastase inhibition by high molecular mass BBI 3-II (Ki = 1.1 x 10(-7) M) have been determined.

Three active-site-acylated derivatives of the activator plasmin-streptokinase complex have been synthesized: n-anisoyl-, n-trans-(N,N,N-trimethylamino)-cinnamoyl- and n-guanidine-benzoyl-plasmin-streptokinase. Their diacylation rate constants were 4.2 x 10(-4), 2.0 x 10(-4) and 0.6 x 10(-4) s-1, respectively. Kinetics of lysis of fibrin clots, containing plasminogen or plasminogen and alpha 2-antiplasmin, by acylplasmin, by a free activator complex and by two acylated activator complexes has been studied. It is shown that in the presence of zymogen and inhibitor the effect of acylactivator, as a fibrinolytic, is 163 times more effective than that of acylenzyme and the fibrinolytic response increases with the doze of acylactivator. The rate of fibrinolysis by a free plasmin-streptokinase complex was higher without the inhibitor than that of fibrinolysis by its acylated derivatives; fibrinolytic action of acylactivators was more effective in the presence of the inhibitor.

In the experiments on guinea-pigs with venous thrombosis there were studied the fibrin- and thrombolytic effects of streptokinase, the plasmin-streptokinase complex and the acylated derivatives of the complex with various rates of reactivation. It was established that the acylated derivatives of the plasmin-streptokinase complex possess greater stability in the blood flow and lead to more prolonged stimulation of fibrinolysis at less magnitude of its systemic activation. Due to this the acylated derivatives of the plasmin-streptokinase complex produce less pronounced fibrinogenolysis. In connection with a high affinity to fibrin their thrombolytic action does not depend on the systemic activation of fibrinolysis.

Kinetics of lysis of fibrin clots from the human, guinea pig, rat and rabbit blood plasma by two active-site-acylated derivatives of the activator plasmin-streptokinase complex with different reaction rate constants has been studied in vitro. It is found that lysis of blood plasma clots in guinea pig is most similar to that of man. Acyl activator dose being increased, the lysis of a plasma clot in guinea pig is accelerated. Two acyl activators exhibit higher fibrinolytic-efficiency as compared to a free activator. Experiments carried out in vivo on guinea pigs with thrombosis show that acyl activators, in contrast to nonmodified plasmin-streptokinase complex induce the less system activation of fibrinolysis and the less fibrinogenolysis.

The interactions of plasmin and plasmin-streptokinase equimolar complex with a number of protein and low-molecular weight substrates as well as protein inhibitors have been examined. It was concluded that the above interactions entail structural changes in the complexes formed, which trigger the activator, increase the rate constants of low molecular weight substrates but create steric hindrances to the interaction with protein substrates and inhibitors.

Kinetics of fibrinolysis by plasmin and plasmin streptokinase complex have been studied using fibrin gels formed from purified fibrin and human blood plasma. The gels were placed into buffer or blood plasma. The contributions of plasminogen and alpha 2-antiplasmin present or absent in both phases to the kinetics of fibrinolysis were quantitatively estimated. In the complex catalyzed fibrinolysis, plasminogen activation reaction dominated whereas in plasmin-catalyzed fibrinolysis, the inhibitor involved reaction, suppressing the process, prevailed.

The binding of the angiotensin-converting enzyme from bovine lung on BrCN-activated Sepharose, CH- and AH-Sepharoses as well as on AH-Sepharose via the carbohydrate fragment of the glycoprotein molecule modulates the possible microenvironment of the enzyme in vivo. It has been shown that the close interaction of the enzyme with the carbohydrate matrix may increase the absolute values of catalytic constants for the hydrolysis of certain substrates. The binding of the angiotensin-converting enzyme to the matrices markedly changes the enzyme activation by chloride ions by causing a shift in the activity optima towards lower activator concentrations.

The angiotensin-converting enzyme from bovine lung was isolated by chromatography with a 25-30% yield and purified 2200-2600-fold. The active molecule concentration in the enzyme preparations was 70-100% as could be judged from titration by inhibitor SQ 20,881. The molecular mass of the enzyme according to electrophoretic data is about 132 kDa; the maximal radius of the enzyme molecule as determined by electron microscopy is 68 +/- 9 A. Five enzyme isoforms with pI of 4.85, 4.7, 4.54, 4.38 and 4.3, respectively, were identified. The kinetic parameters of hydrolysis of three synthetic peptide substrates and the constants of activation of the substrate (Z-Phe-His-Leu) hydrolysis by chloride anions were determined.

A simple and sensitive method has been developed to assess the fibrinolytic activity of plasmin from the change in the column height of fibrin gel. Two conditions were used: 1) 37 degrees C and 16 h incubation at plasmin concentrations of 0.5-50 micrograms/ml and 2) 25 degrees C and 1-2.5 h incubation at plasmin concentrations of 50-1000 micrograms/ml. The method permits to observe the kinetics of fibrinolysis at plasmin concentrations higher that 10 micrograms/ml. The results have shown that the method is applicable for quantitation of plasminogen in human plasma. The method is precise and well reproducible.

Immobilization of alpha-chymotrypsin on carboxymethyl esters of dextran (mol. weights of 60 000 and 100 000), whose macromolecules contain 2-amino-4-chloro-s-triazine residues, results in water-soluble polymeric derivatives of alpha-chymotrypsin, containing up to 60% of the enzyme. The activity of the immobilized enzyme makes up to 90% of the initial one. Using dextran blue as a matrix, a polymeric derivative of alpha-chymotrypsin, containing 9% of a chemically bound enzyme, which retains 20% of its initial activity, has been obtained in a similar way. The thermal stability of the enzyme increases during alpha-chymotrypsin linking to carmoxymethyl esters of dextran. The incorporation of alpha-chymotrypsin into the triacetate cellulose membrane (CTA) during its formation results in a practically complete desorption of the enzyme from the membrane into 1 M NaCl. During incorporation of alpha-chymotrypsin linked to the carboxymethyl ester of dextran (mol. weight 60 000) into the CTA membrane the bulk of the modified enzyme is desorbed, whereas in the case of alpha-chymotrypsin linked to the carboxymethyl ester of dextran with mol. weight of 100 000 or to the blue dextran only an insignificant part of the enzyme is desorbed. The thermal stability of alpha-chymotrypsin and the labile form of the enzyme linked to the carboxymethyl ester of dextran with mol. weight of 100 000 increases during the enzyme incorporation into the CTA membrane.

Experimental data on the rate of enzymatic degradation of a solid protein phase in relation to reagent concentrations in the gelatin gel--proteolytic enzyme solution system was obtained. It was shown that different transformations of the Michaelis-Menten equation do not provide any satisfactory description of the reaction kinetics. A method for the discrimination of putative reaction mechanisms based on the analysis of effective values of constants in the general equation for the rates of dissolution of proteins with different molecular weights was proposed. The experimental results are in favour of a two-step consecutive mechanism taking account of surface concentrations of the adsorbed enzyme and hydrolyzable bonds.

A method of microencapsulating of the proteolytic enzyme alpha-chymotrypsin into semi-permeable nylon membranes is worked out. The membrane is a polimer of 1,6-hexamethylenediamine and sebacoyl chloride. alpha-Chymotrypsin is enclosed into the capsule together with polyethyleneimine, capable of joining the walls of microcapsules and making the membrane more stable. The optimal concentrations of polyenthyleneimine and alpha-chymotrypsin are 5% and 1% correspondingly. The highest yield of microencapsulated enzyme was obtained for completely acetylated delta-chymotrypsin. The kinetic properties of microencapsulated alpha-chymotrypsin change very slightly as compared to those of the native one.

Kinetics of trypsin association with trypsin inhibitor from colostrum (IC) was studied. The association rate constant is 3-10-5 M- minus 1 sec- minus 1 at pH 7,8, 25 degrees C. The rate constant for the complex dissociation was determined from the kinetics of the IC displacement from the complex with trypsin by a specific substrate and was found to be 5-10- minus 6 sec- minus 1 (pH 7,8; 25 degrees C). The equilibrium constant (Ki) was measured in a special experiment and was equal to 4-10- minus 12 M (p H 7,8; 25 degrees C). The similarity of this reaction and the association of trypsin with other protein inhibitors was discussed.

The kinetic parameters of azoalbumin hydrolysis by alkaline proteinase from Bacillus subtilis were determined to be Km = 1.2 . 10(-3) M, kcat = 1.5 sec-1 according to the method of Lainuiwer-Berk and Km = 4 . 10(-3) M, kcat = 0.5 sec-1 from the analysis of the entire kinetic curve. It was found that pH optimum of subtilizin hydrolysis of various substrates and the shape of the curve depended on the substrate nature.

The activation of soluble polysaccharide matrices by titan tetrachloride was studied. A method for obtaining soluble preparations of pancreatic trypsin inhibitor bound to the activated polysaccharide matrix completely retaining its inhibiting activity was developed. It was shown that the equilibrium and kinetic constants for association of modified preparations of pancreatic inhibitor with commercial trypsin did not practically differ from those for native inhibitor. It was found that modification by titan tetrachloride-activated polysaccharides stabilized the protein molecule.

The inhibition by N-benzoyl-L-arginine of subtilisin-catalyzed hydrolysis of various substrates was investigated. Study of combined hydrolysis of these substrates revealed the existence of two productive binding sites in the subtilisin 72 molecule. The type of substrate adsorption depends on the nature of the acyl moiety of the molecule and on the nature of the split-off group. The N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-citrulline methyl ester are bound at the same adsorption site (A), while N-cinnamoyl imidazole and N-acetyl-L-valine methyl ester are found at another adsorption site (B); N-benzoyl-L-alanine methyl ester can be adsorbed both at site A and at site B. However, substitution of the split-off group in the substrates previously adsorbed at site B by the p-nitrophenyl group causes their transfer to site A.

Several procedures for immobilization of basic polyvalent inhibitor of proteases on soluble and insoluble matrices of various chemical nature were studied. Water-soluble high molecular derivatives of the proteases inhibitor were produced, which exhibited the unaltered constants of trypsin inhibition, slower rate of elimination from circulation and the hepatotropism as compared with the native inhibitor. The higher therapeutic efficiency of the high molecular derivative of the proteases inhibitor and of the affinity sorbent, produced on its basis, as compared with the conventional methods of the experimental acute pancreatitis treatment, were confirmed by a number of biochemical data.

The conditions for the microencapsulation of alpha-chymotrypsin into semi-permeable polycarbonate membranes are selected. The diameter of the microcapsules is 100-150 microns. The microencapsulation can be carried out at any value of pH. The effect of the diffusion in the hydrolysis of some esters by microcapsulated alpha-chymotrypsin was estimated. The thermostability of the microencapsulated enzyme was studied.

Modification of pancreatic trypsin inhibitor by carboxymethyl dextran with the aid of carboxyl groups of the carrier activated by water-soluble carbodiimide has been carried out. A special role of electrostatic interactions of protein with the carrier during inhibitor modification has been shown. This modification results in active preparations with high protein concentration on the carrier; their molecular weight is practically equal to that of previously used carrier. A comparison of the thermal stability of high molecular weight preparations of pancreatic inhibitor with respect to protein concentration on the carrier and the number of links between protein and carrier has been carried out.

A theoretical model for transient and steady-state kinetics of microencapsulated enzymes action has been developed. The model is meant to overcome the diffusional limitations, caused by a microcapsulated membrane. The effects of various parameters (enzymatic reaction rate constants, enzyme concentration in microcapsules, membrane permeability, substrate concentration, and bulk pH values) on the overall apparent reaction rate have been analyzed using esters hydrolysis catalyzed by alpha-chymotrypsin encapsulated into polycarbonate membranes as an example.

The microgranules containing covalently bound cis-cinnamoylchymotrypsin were obtained by emulsion polymerization of acrylamide and the cross-linking agent in the presence of a light-sensitive enzyme premodified by acryloylchloride. The kinetics of light reactivation of microgranulated cis-cinnamoylchymotrypsin was studied. It was proposed to develop the latent enzyme image by the reaction of N-acetyl-L-leucine indoxyl ester hydrolysis. The product of this reaction, indoxyl, forms indigo in the presence of oxygen. The intensity of staining was shown to depend on the exposition time. This system can be used for production of half-tone enzyme photomaterials.

Enthalpy of the association of trypsin with pancreatic inhibitor from bovine pancreas at 25 degrees C as a function of pH and ionic strength is estimated. The dependence of the enthalpy on pH is of an extremal character with a minimum at pH 7.6 (delta H degrees =-10.3 ccal/mole). The increase of ionic strength with the addition of LiCl, KCl and CsCl at pH 7.6 leads to be increase of delta H degrees. Possible mechanisms of enthlpy changes depending on medium conditions are considered.

It was shown that protein-protein interactions in microcapsules are possible. A mixture of two proteins at high concentrations was microencapsulated at acidic pH values of the aqueous phase under the following conditions: use of the double emulsification method, use of the inert filler--5% polyethylenimine for the formation of spherical non-aggregated microcapsules containing a small quantity of the protein in the membrane, and use of polycarbonate as a film-forming polymer. The kinetics of trypsin inhibition by soya inhibitor, trypsin autolysis and trypsinogen autoactivation were studied. Encapsulation did not influence the rate constant of the trypsin autolysis reaction and decreased to some extent the specific rate of trypsinogen autoactivation.

alpha-Chymotrypsin and its light-sensitive derivative, cis-cinnamoylchymotrypsin, free and immobilysed (by means of microcapsulation, covalent binding with sepharose 4B and p-aminobenzyl cellulose) are studied. Effect of pH on the hydrolysis rate of N-alpha-acetyl-L-tyrosine ethyl ester by different immobilized enzyme preparations, their acylation with isomeric mixture of cis- and trans-cinnamoylimidazole, reactivation of stable cis-derivatives of different enzyme samples and thermal stability of native and microcapsulated enzyme preparations are investigated.

The present-day concept of the mechanism of interaction of proteolytic enzymes with their natural protein inhibitors is discussed. An extended formal kinetic scheme of the interaction is presented. The applicability of a simplified quantitative characterization of the formation and dissociation of a stable enzyme-inhibitor complex is discussed. A critical consideration is given to the accepted approaches to determination of kinetic and equilibrium parameters of the interaction of model serine proteinases (trypsin and chymotrypsin) with common protein inhibitors of animal and plant origin. Equations are given describing the stationary rate of enzymatic hydrolysis of the respective substrate as a function of the concentrations of the reágents (substrate, inhibitor, enzyme) and kinetic and equilibrium constants. The application of some of the methods is illustrated by the interaction of trypsin and chymotrypsin with the bovine basic pancreatic inhibitor and thermo- and acid-stable inhibitor from rabbit blood serum.

Polyacrylamide beads (microgranules) containing covalently bound alpha-chymotrypsin were obtained. An average diameter of the microgranules is 100--150 micrometers. Prior to the bead polymerization alpha-chymotrypsin was modified by acryloyl chloride. The efficiency of the microgranulated alpha-chymotrypsin action depends on the kinetic properties of the substrates used. The pH optimum for the microgranulated enzyme activity towards N-acetyl-L-tyrosine ethyl ester is shifted by 1,5 units to the alkaline zone. The immobilized alpha-chymotrypsin is completely stable for 100 hrs at 50 degrees.

A method of isolating preparations of pancreatic inhibitor of trypsin, bound with soluble polysaccharide carriers, is worked out. It is demonstrated that the reaction of a pancreatic inhibitor and cyanuric chloride-activated dextran proceeds for OH groups of tyrosine residues and for-epsilon-NH2 groups of lysine residues. A method is offered of the protection of amino groups with citraconic anhydride for the complete retaining of the inhibitory activity during attachment to dextran. Thermic denaturation of pancreatic inhibitor preparations at pH 4.7 and 97 degrees C is studied. It is found that the modification by 2-amino-4.6-dichloro-s-triazine stabilizes the protein molecule, while the interaction with the matrix of soluble dextran does not carry any contribution to thermostability of the pancreatic inhibitor.

The effects of electrostatic charge of the matrix on the pH-dependence of interactions of commercial trypsin with preparations of pancreatic inhibitor modified by soluble polysaccharide coupling were studied. It was shown that the rate constants of trypsin association with native and modified pancreatic inhibitor preparations as well as the rate constants of dissociation of their complexes and, consequently, the inhibition constants are identical. The invariability of the rate constants for the association reaction after the increase in the molecular weight of pancreatic inhibitor may be probably accounted for by the fact that the limiting step of a stable trypsin-inhibitor complex formation is not controlled by diffusion. Thermal denaturation of pancreatic inhibitor preparations modified by binding to polysaccharides (pH 4.7--8.0, 97 degrees C) suggests an essential role of the negative charge of matrix in stabilization of the protein inhibitor globule.

Plasmin, free of an activator, was obtained after activation of the highly purified human plasminogen by means of trypsin immobilized on Sepharose 4B and after removal of the enzyme from the system. Plasmin was acylated at the active centre using p-amidinophenyl ester of p'-anisic acid to produce the fibrinolytic agent causing a long-term effect. Inactive acyl plasmin was deacylated with the constant rate k3 = 3.10(-4) c-1 (r1/2 = 40 min) at pH 7.4 and 37 degrees. Preliminary studies of the anisoyl-plasmin effect on the system of fibrinolysis in rats showed that in the absence of fibrin clot the fibrinolytic activity was not increased immediately in the euglobulin fraction of blood but after 30 min in result of deacylation of the acyl-enzyme administered into circulation.