[Phenothiazine derivatives as enhancers of peroxidase-dependent chemiluminescence].

Some N-alkyl phenothiazines with different ionic groups were studied as enhancers of chemiluminescence catalyzed by soybean peroxidase. Phenothiazines carrying positive charged groups had no an enhancement ability, whereas phenothiazine derivatives with negative charged groups increased significantly an intensity of chemiluminescence. Correlation between the enhancement activity ofphenothiazines and their ability to enzymatically oxidize by hydrogen peroxide was found. The discovery of new enhancers open good perspectives for an improvement of sensitivity of analytes determination by chemiluminescent enzyme immunoassay.

[Enzyme immunoassay for the determination of hexestrol in meat].

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an antibiotic forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20-IC80) equaled 0.03-0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05 ng/ml, 2.9 ng/ml, and 0.26-32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.

[Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig].

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Enzyme detection limit in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.

[Use of soybean peroxidase for the enzyme immunoassay of sulfamethoxipyridazine in milk].

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3-63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 microg/l) by several orders of magnitude.

[Oxidation of luminol with peroxidase from royal palm leaves].

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.

[Oxidase-peroxidase method for determination of ethanol in fermented musts and wine products].

A new alcohol oxidase-peroxidase method of determination of ethanol content in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). This method is less time- and labor-consuming and allows simultaneous testing a series of wine samples.

[Enzymatic synthesis of a conducting complex of polyaniline and poly(2-arcylamido-2-methyl-1-propanesulfonic ACID) using palm tree peroxidase and its properties].

An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.

[Experimental development of a method of enzyme therapy in eye burns]. / Eksperimental'naia razrabotka metoda énzimoterapii ozhogovoi bolezni glaz.

The proposed method of enzyme therapy of eye burns consists in instillations of a proteolytic drug for the first two days after burn and instillations of an antiproteolytic agent for the next 12 days. Experimental trials of the method in a rabbit with an alkaline burn of the cornea, including assessment of clinical manifestations of disease and investigation of the proteinase-inhibitor balance in the lacrimal fluid demonstrated a high efficacy of the new treatment.

[Pharmacokinetic study of collagenase from the king crab, Paralithodes camtschatica]. / Farmakokineticheskoe izuchenie kollagenazy kamchatskogo kraba Paralithodes camtschatica.

The king crab collagenase was shown to permeate through both aseptic and purulent wounds into rat circulation. The rate of enzyme permeation was higher in purulent wounds, while the enzymatic activity did not affect the rate of collagenase transport into blood. After intravenous administration the enzyme was mainly accumulated in liver and kidney, while protease was not found in the heart, spleen, lung and muscle tissues. Collagenase was rapidly eliminated from circulation and its isozymes were contained in blood not in free state, but they were bound and inactive.

[Stability of isoenzyme C of the collagenolytic protease from the crab Paralithodes camtschatica]. / Stabil'nost' izofermenta s kollagenoliticheskoi proteazy kraba Paralithodes camtschatica.

The stability of the isozyme C of the collagenolytic protease of the king crab (Paralithodes camtschatica) has been studied. It has been shown that protease C is stable at mild alkaline conditions; marked inactivation is observed only at temperatures above 40 degrees C. Under these conditions the enzyme thermoinactivation is a monomolecular process. CaCl2 and NaCl have no effect on the thermoinactivation process. At pH < 6 the enzyme is rapidly and irreversibly inactivated, presumably due to the autolytic cleavage. At acidic conditions the enzyme is strongly stabilized in the presence of CaCl2, this effect being critically dependent on the salt concentration. A stabilizing effect is also observed by an increase in the protease C concentration. The enzyme is the most labile in strongly alkaline condition; the process its inactivation at pH 11.25 is not dependent on the enzyme concentration. The protease C retains its activity in the presence of detergents but is effectively inhibited by guanidine chloride.

[Hydrolysis of proteins by collagenolytic proteinases from the king crab]. / Gidroliz belkov kollagenoliticheskimi proteinazami kamchatskogo kraba.

Hydrolysis of collagen molecules in the presence of collagenolytic proteases A and C from the king crab has been studied by electrophoresis. Both proteases are shown to hydrolyze effectively type I and III collagens, patterns of the products differing for the proteases A and C. The thermal denaturation of the type I collagen increased the effectiveness of the enzymatic hydrolysis. The crab collagenolytic proteases catalyze the hydrolysis of such proteins as bovine serum albumin, ovalbumin, horse cytochrome c, mouse immunoglobulin G, casein and human fibrinogen, only elastin being resistant. The mechanisms of the fibrinogen cleavage differ not only for the proteases A and C but also for plasmin, whereas the efficiencies in all the cases are similar.

[The immunohistochemical study of suppurative wounds in rats following the application of collagenase from the crab Paralithodes camtschatica]. / Immunogistokhimicheskoe izuchenie gnoinykh ran u krys posle applikatsii kollagenazy kraba Paralithodes camtschatica.

Immunohistochemical study of tissues of purulent wounds in rats after application of the collagenase isolated from the king crab Paralithodes camtschatica has been undertaken. The enzyme therapy resulted in a rapid and efficient removal of necrotic debris. It was accompanied by fibrin elimination from the wound bottom and subsequent formation of new capillaries. Cellular fibronectin with ED-A sequence was identified in the newly formed granulation tissue, which points to its active synthesis in situ. Detection of type I collagen in granulation tissue revealed that wound treatment with crab collagenase had no impact on the development process of the tissue. Polyclonal antibodies against two isozymes of crab collagenolytic protease were obtained. It was shown that after application of both isozymes of the collagenase were accumulated in fibrin deposits at the wound bottom but not penetrated in adherent granulation tissue. These processes underlie the therapeutic effect of the crab collagenase.

[Elastase from the hepatopancreas of the king crab]. / Elastaza iz gepatopankreasa kamchatskogo kraba.

Using ion-exchange chromatography on DEAE-Sepharose and gel-filtration on Sephacryl S-200, a homogeneous preparation of elastase with a specific activity towards Boc-(Ala)3-pNA of 3.7 u./mg has been isolated from the hepatopancreas of the king crab Paralithodes camtschatica. The molecular mass and isoelectric point for the enzyme are equal to 28500 Da and 4.5, respectively. The amino acid composition of the isolated protease has been established. Analysis of catalytic properties of the enzyme revealed that the maximal elastase activity is observed at pH 8.0-8.5; the Km and kcat values for Suc-(Ala)3-pNA are 4 mM and 3 s-1, respectively. The enzyme activity is fully inhibited by elastinal and diisopropylfluorophosphate, but not by N-ethylmaleimide, 2-mercaptoethanol, p-chloromercuribenzoate, EDTA, or o-phenanthroline, which makes it possible to refer the enzyme to the class of serine elastases. The activating effect of crab elastase was demonstrated in the presence of inorganic salts. The elastase is stable in neutral and alkaline solutions containing detergents as well as at temperatures below 45 degrees C (pH 8.0).

[Changes in the level of the angiotensin-converting enzyme activity in the spermatozoa of patients with chronic prostatitis and of participants in the cleanup of the accident at the Chernobyl Atomic Electric Power Station]. / Izmenenie urovnia aktivnosti angiotenzin-prevrashchaiushchego fermenta v spermatozoidakh patsientov s khronicheskim prostatitom i u uchastnikov likvidatsii avarii na Chernobyl'skoi AES.

The method of determination of angiotensin-converting enzyme activity in human spermatozoa and sperm plasma was developed. The amount of total and active-mobile spermatozoa was shown to be less in Chernobyl victims than in healthy donors. Moreover, the specific activity of angiotensin-converting enzyme in Chernobyl victims spermatozoa calculated per 10(6) cells was 12-fold greater than in spermatozoa of the donors. Similar phenomenon, although to a les extent, was observed in patients with chronic prostatitis. The enzyme activity in blood serum and sperm plasma was also demonstrated to be similar for all groups investigated and equal to that in healthy donors.

[A morphological evaluation of the action of collagenase from the king crab Paralithodes camtschatica on the experimental wound process]. / Morfologicheskaia otsenka vozdeistviia kollagenazy kamchatskogo kraba Paralithodes camtschatica na ranevoi protsess v éksperimente.

The paper presents the results of experimental morphological evaluation of the effect of a new proteolytic enzyme collagenase of crab Paralithodes camtschatica on wound healing in infected and aseptic rabbit wounds. The enzyme was applied on wounds using gauze and gelevin. The findings show that this protease is highly effective for debridement of infected wounds, the effect increasing at gelevin addition. To reach maximal therapeutical effect and diminish the inhibition effect of collagenase on granulation tissue, it is recommended to reduce the dose of protease during debridement process. Clinical application of crab collagenase must be individual as well as duration of wound enzymotherapy.