New Sorbent on the Basis of Covalently Immobilized Lysozyme for Removal of Bacterial Lipopolysaccharide (Endotoxin) from Biological Fluids.

It was demonstrated for the first time that immobilized lysozyme can efficiently remove Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (endotoxins) from solutions. Experimentally confirmed sorption capacity for the developed sorbent was at least 400 ng of endotoxin per ml sorbent. The new sorbent is compatible with the whole human blood and can be potentially used in extracorporeal therapy in the treatment of sepsis.

[The study of protease primary specificity by statistical analysis of MALDI mass-spectra of proteolysis products].

Experimental verification for studying of proteolytic enzymes' primary specificity by statistical analysis of MALDI (matrix-assisted laser desorption/ionization) mass spectra of products obtained by protein substrates proteolysis was done by the use of proteinases with known substrate specificity (trypsin and glutamylendopeptidase). Proposed technique not requires direct determination of proteolysis products amino acid sequences, reliably establishes proteinases with anarrow substrate specificity and shows a relative tolerance for the presence in MALDI mass-spectra peaks of contaminants. It was shown that the pseudo-positive results exception requires the use of protein substrates series with the following averaging received statistical data.

[Theoretical possibilities and limitations of protease primary specificity determination by statistical analysis of MALDI mass-spectra of proteolysis products].

Possibilities and limitations of method examination of proteolytic enzymes' primary specificity by statistical analysis of MALDI (matrix-assisted laser desorption/ionization) mass spectra of products obtained by protein substrates proteolysis without direct determination of their amino acid sequences were investigated theoretically. The optimum ranges given by the errors of the peptides masses measuring for the fabrication of statistical set of the events and the form of statistical data presentation were chosen. It was shown that the proposed method can be applied only for proteases with a relatively narrow primary specificity (two or three amino acids). The influence of protein substrate molecular weight and amino acid composition on the efficiency of specific to a particular protease amino acids display under statistical treatment of the set of proteolysis products masses was studied on the model of trypsin, chymotrypsin, glutamylendopeptidase, pepsin (pH 1.3).

[Enzymatic hydrolysis of keratin-containing stock for obtaining protein hydrolysates].

Optimization of the process of enzymatic hydrolysis of keratin-containing stock aimed at obtaining hydrolysates of high biological value has been performed. The increasing of the stock/water weight ratio, the amount of the alkaline protease preparation from Acremonium chrysogenium added and the temperature of the reaction mixture resulted in an increase in the yield and antioxidant capacity of hydrolysis products. The molecular masses of soluble products obtained under optimal hydrolysis conditions ranged from 3.55 to 3.60 kDa. High antioxidant capacity, 100% bioavailability and a well-balanced amino acid composition was characteristic of the hydrolysis products.

[A mass-spectrometric approach to primary screening of collagenolytic enzymes].

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestesfrischi and D. maculates) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks ("fingerprint") characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.

Enzyme activity regulation by an outer physical signal.

The main principles and methods of creating artificial systems with the directional regulation of enzyme activity by an outer physical signal are discussed. We are presenting some experimental results for the systems working in the necessary regime and responding to light and temperature.

Enzyme-dependent responses of stimuli-sensitive systems.

The methods of creating of systems where properties depend both on an outer physical signal and enzyme activity are discussed. The construction of a conjugation chain between responses of stimuli-sensitive materials and urea hydrolysis catalyzed with urease is demonstrated on the basis of light- and thermoreversible polymeric matrices.

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis.

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized urease actually has the same catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.

[Interaction of alpha-chymotrypsin with dimethylsulfoxide: a change of substrate could change the mechanism of interaction]. / Vzaimodeistvie alpha-khimotripsina s dimetilsul'foksidom: smeni substrat--"izmenish'" mekhanizm vzaimodeistvii.

The kinetic behavior of alpha-chymotrypsin was studied in water-DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with alpha-chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S2-site of substrate binding to the active site of the enzyme.

[Phase transition in the matrix as a regulator of enzymatic activity of proteinases]. / Fazovyi perekhod v matritse kak reguliator fermentativnoi aktivnosti proteinaz.

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.

[Anomalous temperature dependence of the activity of immobilized alpha-chymotrypsin preparations]. / Anomal'naia temperaturnaia zavisimost' aktivnosti preparatov immobilizovannogo alpha-khimotripsina.

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.

[Fibrinolysis in vitro by acylated derivatives of a plasmin-streptokinase activator complex]. / Fibrinoliz in vitro atsilirovannymi proizvodnymi aktivatornogo kompleksa plazmin-streptokinaza.

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.

[Effectiveness of acylated thrombolytic agents in lysis of blood plasma clots from humans and various animals]. / Effektivnost' atilirovannykh tromboliticheskikh agentov v lizise sgustkov iz plazmy krovi cheloveka i zhivotnykh razlichnykh vidov.

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.

[Kinetics of in vitro fibrinolysis by plasmin and a plasmin-streptokinase equimolar complex]. / Kinetika fibrinoliza in vitro plazminom i ékvimoliarnym kompleksom plazmin-streptokinaza.

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.

[Kinetics of dissolution of solid protein substrates by proteinases. Selection of the reaction mechanism]. / Kinetika rastvoreniia tverdykh belkovykh substratov proteinazami. Vybor mekhanizma reaktsii.

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.

On the mechanism of interaction of organic solvents with the active site of alpha-chymotrypsin.

Kinetic behavior of alpha-chymotrypsinin the reaction of hydrolysis of the N-acetyl-L-tyrosine derivatives was investigated in non-denaturing water-dimethylsulfoxide and water-ethanol mixtures. Similar specific interactions between the two solvents and the active site of alpha-chymotrypsin were shown to result in similar kinetic effects. It is proposed that the changes in the active site structure of the enzyme caused by the interaction with the organic solvents ("conformational isomer" formation) resulted in two parallel processes--acceleration of the acyl-enzyme formation step and a decrease in the deacylation rate. The possible molecular mechanism of this phenomenon and an adequate kinetic model describing the data are discussed.

[A method for determining plasmin by the rate of fibrin gel lysis]. / Metod opredeleniia plazmina po skorosti lizisa fibrinovogo gelia.

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.

Relationship between state of a thermosensitive matrix and the activity of urease immobilized in it.

The effect of temperature on kinetic and equilibrium parameters of urea hydrolysis catalyzed with urease immobilized into a thermosensitive poly-N-isopropylacrylamide gel was studied. The temperature behavior of the gel-urease system is different from similar systems. After a decrease in the enzyme activity above the critical temperature, the maximal rate of the enzymatic reaction and gel swelling ratio begin to increase. Urea hydrolysis catalyzed with immobilized urease and shrinking-swelling of the thermosensitive urease-containing gel depend on each other. Under collapse, gel swelling increases due to the enzymatic reaction. The rate of the enzymatic reaction no longer follows Michaelis-Menten kinetics, and the dependence of the reaction rate on substrate concentration becomes more complicated.

[Microgranulated form of cis-cinnamoylchymotrypsin for half-tone enzyme photography]. / Mikrogranulirovannaia forma tsis-tsinnamoilkhimotripsina dlia polutonovoi fermentnoi fotografii.

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.

[Properties of alpha-chymotrypsin, covalently incorporated into polyacrylamide microgranules]. / Svoistva al'fa-khimotripsina, kovalentno vkliuchennogo v sferncheskie mikrogranuly iz poliakrilamida.

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.