Search

The study of antimicrobial compounds of animal origin, particularly antimicrobial peptides (AMPs), is a current research topic. However, extracting endogenous AMPs is a challenging process and requires the application of targeted enzymatic processing principles based on knowledge of the structure of prepropeptide molecules — precursors of AMPs. In this study, a search was conducted for antimicrobial peptides present in Sus scrofa and Bos taurus organisms, as well as their precursors, using The Antimicrobial Peptide Database and UniProtKB databases. In the amino acid sequences of prepropeptides, the sequences of the mature peptides were found, and cleavage sites for trypsin, bacterial collagenase (type I), and neutrophil elastase were determined. As a result of the search for antimicrobial compounds in The Antimicrobial Peptide Database, 18 antimicrobial peptides from Sus scrofa and 40 antimicrobial peptides from Bos taurus were identified. Based on the results of determining cleavage sites in AMP precursors, enzymes were ranked from less preferred to more preferred for AMP release as follows: bacterial collagenase (type I) ≤ trypsin < neutrophil elastase. This order is justified not only by the number of suitable cleavage sites and their accuracy but also by the action of enzymes within mature AMPs: it is important to consider that enzymes can “cut” the peptides themselves, thereby reducing their antimicrobial activity. The bioinformatics analysis conducted is applicable for both primary screening of raw material potential and determining of suitable enzymes for extracting antimicrobial compounds from Sus scrofa and Bos taurus organisms.

SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves mea­suring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simulta­neously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with ¹⁸O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H₂¹⁸O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For ¹⁸O labelling, the authors dried the peptide and incubated it in H₂¹⁸О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with ¹⁸O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corre­sponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with ¹⁸O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

Chicken eggs proteins and their derivatives, like protein hydrolysates, peptides and amino acids, possess high nutritional value and provide a wide range of biological activity. They serve as sources for development of functional ingredients that draw the attention of specialists in the food production and biomedical industries, as well as the livestock feed industry. Enzymatic hydrolysis of proteins is a popular process for obtaining bioactive peptides with multifunctional properties. The purpose of this study is to obtain a hydrolysate of ovalbumin with a high degree of hydrolysis and to determine its functional and technological parameters. The research presents a two-stage scheme of ovalbumin hydrolysis with the help of pepsin and trypsin which provide high degree of hydrolysis (82–83%). The fractional composition of the hydrolysate is determined. The fractional composition is represented by three main fractions (high, medium and low molecular weight). The summarized antioxidant activity (SAA) of the hydrolysate is considered within the dynamics of the hydrolysis process. The highest SAA value was noted after 2 hours, and it amounted to 170.23 mg/l; at the end of hydrolysis the SAA value was equal to 114.31 mg/l. When analyzing the SAA, it was found that the main contribution to the summarized antioxidant activity of the ovalbumin hydrolysate is made by peptides of the medium molecular fraction. The microfiltration process, used in the research, made it possible to separate high-molecular compounds, which led to an increase in the SAA of the hydrolysate to 189.9 mg/l. The main functional and technological parameters of the hydrolysate are determined in this research. The comprehensive study of the biological activity and functional characteristics of egg protein hydrolysates and their peptides provides a theoretical basis for expanding the range of functional ingredients obtained from food proteins and for replenishing the range of functional foods.

Soy continues to be one of the top sources of vegetable protein. Structurally modified soy proteins and processed products are used as part of functional foods. Enzymatic hydrolysates of food proteins have different degrees of hydrolysis and functional profiles, hence the constant search for the optimal hydrolysis parameters. The present research objective was to design a two-stage enzymatic conversion process of soy protein using mathematical methods, as well as to evaluate the antioxidant properties of the hydrolysate in laboratory conditions. Soy protein isolate was tested to define the maximal value of the hydrolysis degree. It underwent a series of two-factor experiments in the presence of pepsin and trypsin. The study focused on the hydrolysis time and the enzyme-substrate ratio. The results were optimized using the response surface methodology in MathCad 15. The total antioxidant activity of the hydrolysate during hydrolysis was determined on a Tsvet-Yauza-01-AA chromatograph using the amperometric method. For the pepsin test, the processing time was 7 h and the enzyme-to-substrate ratio was 1:22. For the trypsin test, the time was 7 h and the ratio was 1:30. The mathematical modeling revealed the following optimal parameters. The first stage involved hydrolysis with pepsin for 5 h at an enzyme-to-substrate ratio of 1:20; the second stage involved hydrolysis with trypsin for 3 h at an enzyme-to-substrate ratio of 1:19. The resulting hydrolysate demonstrated 88% hydrolysis. The highest summary antioxidant activity was registered after 5 h of hydrolysis and amounted to about 250 mg/100 mL. The resulting enzymatic hydrolysate of soy protein can be used as a food component or an antioxidant feed additive. The obtained peptides can immobilize essential microelements, e.g., Zn, I, and Se, as well as produce polyvalent complexes. Further studies will be aimed at the residual antigenicity of the hydrolysate and other functional indicators.

Relevance. An increase in the incidence of whooping cough, a high proportion of severe forms of the disease, and a decrease in the sensitivity of circulating strains of B. pertussis to antibiotics require the development of more effective etiotropic therapies, including those capable of influencing biofilm forms of the whooping cough pathogen, which differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs.Аim of the work is to study the effect of trypsin and lidase in combination with gentamycin on the growth of biofilms of Bordetella pertussis strains on an abiotic substrate.Materials and methods. In the experiments B. pertussis strains isolated in the Russian Federation from whooping cough patients in 2001‒2010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin (10 mcg/ml), lidase (20 IU/ml), gentamycin (2.0 mg/ml, 0.4 mg/ml and 0.08 mg/ml) and their combinations in roundbottomed polystyrene 96­well plates was evaluated by staining with 0.1% gentian­violet solution.Results. Gentamycin partially suppressed the formation of biofilms and caused partial destruction of the formed biofilms in the absence of growth of microbial colonies when sowing supernatants from biofilm cultures on a dense nutrient medium. The minimum suppressive concentration of gentamycin (MSC) was 2 mg/ml. Trypsin completely suppressed the growth of biofilms and caused the complete destruction of the formed biofilms. Lidase also suppressed the growth of biofilms, but less effectively affected the formed biofilms. The growth of colonies typical of B. pertussis was noted when sowing supernatants from biofilm cultures in the presence of trypsin and lidasе on a dense nutrient medium. Trypsin in combination with all the studied concentrations of gentamycin completely suppressed the growth of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused complete destruction of biofilms in the absence of microbial growth on a dense nutrient medium. Lidase in combination with all the studied concentrations of gentamycin also suppressed the formation of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused partial destruction of the formed biofilms in the absence of microbial growth on a dense nutrient medium.Conclusion. The synergistic effect of the combination of trypsin and lidase with gentamycin on growing and formed biofilms of B. pertussis strains was revealed. The combined use of trypsin or lidase with gentamicin reduced its MSC for growing biofilms by 25 times. The most pronounced effect on the formed biofilms was the combination of trypsin with gentamycin at a concentration of 2 mg/ml, which caused their complete destruction and death of planktonic cells. The effect of the combination of lidase with gentamycin on the formed biofilms was less pronounced.

Aim. Study the effect of trypsin, lidase (hyaluronidase) and fluimucil (N-acetyl-L-cysteine) on the growth of biofilms of Bordetella pertussis strains on the abiotic substrate. Materials and methods. In the experiments, the strains of the main B. pertussis serotypes isolated in the Russian Federation from whooping cough patients in 20012010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin, lidase and fluimucil in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. Results. Trypsin suppressed the growth of biofilms and destroyed the formed biofilms. Lidase suppressed the growth of biofilms less actively, without affecting the formed biofilms. Fluimucil did not affect both the growth of biofilms and the formed biofilms. The growth of colonies typical for B. pertussis was noted when planting fluids from cultures in the presence of preparations, as well as from culture control wells on a dense nutrient medium. Conclusion. The different effect of the drugs studied by us may be related to the different quantitative content of targets for trypsin (proteins), lidase (mucopolysaccharides, containing uronic acids), fluimucil (acid mucopolysaccharides) in the biofilm matrix. The growth of the typical morphological properties of the colony of B. pertussis during the sowing of culture seedlings on a dense nutrient medium testifies to the destruction of the biofilm matrix by trypsin and lidase in the absence of influence on plankton cells.

The review focuses on the role of neutrophilic extracellular traps (NETs) in systemic complications of acute pancreatitis. NETs can activate trypsin, cause inflammation and pancreatic tissue damage, and clog the excretory ducts. The main fatal complications of acute pancreatitis, such as acute lung injury, kidney, myocardial and CNS damage, intestinal dysfunction, hemocoagulation disorders are associated with NETs. Focusing on the formation and degradation of NETs may be a way to develop strategies for treating organ damage in severe acute pancreatitis. Current data on the use of NET-targeted therapy in experimental severe acute pancreatitis, which is aimed at blocking the NETs formation and disassembly of the DNA scaffold, inhibition of proteins toxicity in NETs, are considered. [ABSTRACT FROM AUTHOR]

Introduction. Public health is gradually deteriorating as a result of unhealthy lifestyle and diet, which triggers allergic reaction to certain foods. Milk and dairy products are rich in biologically active substances, which makes them a good dietary supplement for athletes, diabetic patients, etc. However, this popular food contains allergens, for instance, such proteins as αS1-casein, αS2-casein, β-casein, and κ-casein. Therefore, one of the most urgent tasks of modern food science is to reduce the allergenic properties of casein. Heat treatment is an option, but thermal exposure leads to denaturation and produces new antigenic determinants, e.g. epitopes. Biotechnological processing is a more promising method. It is based on the catalytic properties of proteolytic enzymes. Enzymes make it possible to obtain a protein hydrolyzate with amino acids of various molecular weights. The present research provided the optimal working parameters of casein proteolysis by various enzymes (endopectidases), namely trypsin, chymotrypsin, and thermolysin. Study objects and methods. Casein hydrolysates are casein-based biopeptides, and casein is an accessible and valuable milk protein. Trypsin, chymotrypsin, and thermolysin were used as proteases. The experiment was based on standard methods. Results and its discussion. At 47 ± 2°C and pH 7.5 ± 0.2, the production of low-molecular-weight components of casein hydrolyzate proved feasible when thermolysin was used at a ratio of 1:100 for 24.00 ± 0.05 h, and chymotrypsin and trypsin – at a ratio of 1:25 for 24.00 ± 0.05 h. Conclusion. The resulting casein hydrolysates contain biologically active peptides and can be used in formulations of low-allergy functional dairy products in allergy-friendly, sports, and baby diets.

A mandatory requirement for cell cultures used in scientific researches and biomanufacturing is the absence of their contamination by viruses. We have described the case of adventitious isolation of mammalian orthoreovirus in the rhesus macaque embryo kidney cells MA-104. PCR analysis for the presence of reovirus RNA of all probable sources of reovirus (trypsin, fetal bovine serum, clinical samples, cell culture) revealed no viral RNA in any of the samples. An important condition for the activation of the reovirus reproduction in the MA-104 cells was the presence of trypsin in the culture medium. The obtained results underscore the urgency of control for the reovirus contamination of chemicals of animal origin and cell cultures. Since reoviruses are associated with human diseases, such control in pharmaceutical production is mandatory.

Trypsin is a reagent widely used in the manufacture of biological medicinal products (BMPs). Until recently, pancreata of cattle, pigs and poultry were the main sources of trypsin preparations. The discovery of the disease called «transmissive spongiform encephalopathy» or «cow rabies» (TSE) in cattle in the late 1980s showed a clear need for limiting the use of this source. Given the potential risk of using trypsin obtained from cattle, porcine trypsin became more commonly used in the production of biological medicinal products. Enzymes obtained from raw materials of animal origin can be contaminated with circoviruses, parvo- and pestiviruses, and mycoplasmas that are common to pigs. Due to high resistance to physical and chemical treatment, these contaminants pose a potential risk to recipients of vaccines, as well as to other biological medicinal products. Prevention of contamination requires measures aimed at detection, reduction and inactivation of foreign agents, both in raw materials and during BMP production. The article considers the most common types of porcine trypsin contamination, methods of its detection, reduction and elimination. The article also contains information on the Russian and international requirements for the quality and safety of porcine trypsin used in the production of biological medicinal products.

This study is dedicated to trypsin – carbon nanotube hybrid materials, and the possibility of their use in biosensor devices. The morphology of the enzyme – nanotube materials have been studied with AFM microscopy. The volt-ampere characteristics of the materials have been measured with potentiometric methods. It has been shown that the enzyme interacts with the carbon nanotube in the solution by covering its surface and creation of a hybrid material without tis inactivation. The pure enzyme on the surface of mica creates small peaks protruding from the porous surface of the substrate. They have a height of 6 to 7 nm. The quaternary globular structures of the enzyme is visible. When creating hybrid materials using trypsin, carbon nanotubes play an important role, which radically change the adhesion of the enzyme to the surface, which leads to the appearance of isolated clusters of a hybrid material. The surface of nanotubes is completely covered by the enzyme. In this case, structures with a maximum height of 9 nm to 11 nm are formed. It has been shown that the voltampere characteristic of the resulting hybrid material is sensitive to the presence of casein. The curves of the I-V characteristic have characteristic peaks in the range of 500 and 700 microvolts. The tests with electrodes modifi ed with hybrid materials showed better response times than electrodes modifi ed with pure trypsin. It is likely that hybrid materials contribute to the linearity of the voltmetrometric characteristics when detecting the casein. It has been shown that the screen-printed electrodes modifi ed by trypsin and carbon nanotubes are promising in use in the food industry to detect casein in raw materials.

The production of biological medicinal products used for prophylactic, diagnostic and therapeutic purposes includes the use of inherently variable biological processes and materials. In order to ensure the quality of the finished biological product it is necessary to determine the source, nature and fitness for use of the starting materials, including reagents, culture media, buffer solutions, sera, and enzymes. The article summarises literature data on the structure, properties and mode of action of the animal-derived reagent trypsin — proteolytic enzyme widely used in the production of biological medicinal products. The article dwells upon the sources of trypsin, methods of its production, requirements for trypsin products of different purity grades intended for human and veterinary use as well as for use as reagents in the production of vaccines, advanced therapy medicinal products and genetically engineered products. The article describes the role that trypsin plays in the human and animal intestinal digestive enzyme systems, in dissociation of cells in cultures in the process of cell passaging, in the mechanism of proteolitic activation and inactivation of a wide range of viruses, and in the examination of proteins primary structure.

The comparative experiments demonstrated strong correlation between the index of Yersinia pestis EV NIIEG strain adhesion to human red blood cells and hydrophobic properties of bacteria. At the same time no dependence between hemagglutination and adhesion of plague microbe was determined. Bacterial culture treatment with trypsin and high temperature (56 °C) decreased adhesion. Incubation of microbial cells with red blood cells in the presence of antibiotics (ampicillin, gentamycin, doxycycline and cefotaxime) had no influence on their interaction. The results of investigations suggested the major role of hydrophobic interactions in Y.pestis EV NIIEG strain adhesion to human red blood cells provided by the surface structures of microbial cells of the lipoprotein origin.

The paper discusses some aspects of environmental safety, one of which is the activity of trypsin inhibitors plant material class oxidases enzymes and methods of inactivation of these factors antinutritive. A comparative study of thermal inactivation method of the compounds under the action of electric discharges with nanosecond rectangular front. [ABSTRACT FROM AUTHOR]

The study objective is to develop approaches to the retrospective assay of tropicamide in biological fluids and hair. The study was performed using the substance of tropicamide. Sample preparation included hydrolysis with the following enzymes: papain, chymotrypsin, trypsin, chymopsin, and hyaluronidase. Extracts were analyzed using a gas chromatograph with mass selective detection Technologies (USA) 7890 A/5977 MSD. When modeling long-term use of tropicamide, male rats of white and brown natural color, about 6 months old and weighing 200-250 g, were used. Animals were injected with a tropicamide solution in the tail vein for 28 days at a dose of 40 mg/kg of body weight. After 28 days of administration of the tropicamide solution, daily urine and blood were collected, and hair was cut from the back and sides of the animal's body. After another 28 days, hair samples were taken again. Within the first 6 hours after the last tropicamide dose, its blood concentration reached the maximum (191.6 μg/ml) and within 4 days decreased by 10 times; in the urine, within the first 24 hours, tropicamide level decreased from 627.7 to 489.9 μg/ml, then for 2-3 days it remained approximately at the same level. From day 4, the tropicamide concentration significantly decreased, and on days 11-12, it was not detected in the urine. After 4 weeks, the tropicamide content in the hair was at the level of quantification (1.25-2.20 ng/mg) and could be detected only by sample preparation by enzymatic hydrolysis with papain. Thus, the developed and validated methods for the enzymatic hydrolysis of biological fluids and hair allowed retrospective studies of biological fluids and hair with high reliability.

The objective of the present study was the development of the method for the extraction of toxic compounds from the uncoloured hairs with the use of enzymatic hydrolysis by proteolytic enzymes of the toxic compounds comprising the group of pharmaceutical substances present on the uncoloured hairs collected for the forensic medical expertise. The experiments were carried out using laboratory animals, viz. guinea pigs having hair of natural white and black colour and white rats that were given per os a phenobarbital solution and a dimedrol solution as well as daily intravenous injections of tropicamide hydrochloride administered every 28 days. The hair obtained from the experimental animals was subjected to acidic, alkaline, and enzymatic hydrolysis with chymotrypsin, trypsin, and papain. The analysis of the extracted materials was performed using the gas chromatographic technique with mass-selective detection. The results of the study give evidence that the completeness of the extraction of the toxic substances from animal hairs with the use of enzymatic hydrolysis is twice and thrice that of acidic and alkaline hydrolysis respectively. The enzymatic hydrolysis methods developed in the present study are equally efficient using the naturally coloured white and black hairs of the laboratory animals which allows to recommend them for the extraction of poisonous substances from such material. The methods were validated.

MDCK and Vero (B) cell cultures were propagated during 10 passages in the experimental nutrient media containing the soybean powder hydrolyzate prepared using trypsin and bromelain enzymes and the rice powder hydrolysate prepared with trypsin and in the control DMEM and SFM4 MegaVir media. The karyological, morphological, and proliferative characteristics of continuous cultures were examined and compared. The experimental media supplied with 3% fetal bovine serum (FBS) (Gibco, U.S.A.) showed high growth-enhancing properties and failed to affect their morphology. After propagated during 10 passages in the experimental media preserved a stable karyotype. MDCK cell cultures in the nutrient media based on rice and soybean powder hydrolyzates low (2%) in FBS caused no substantial changes in the proliferation indices and morphological and karyological characteristics of cell cultures.

Comparative reproduction studies of 7 avian influenza virus strains (H5N1, H5N2, H3N2, H4N6, H7N7) in Vero and MDCK cell lines have indicated that the MDCK cell line is an optimal substrate for all study strains. The maximum viral output depends on trypsin concentrations and infection doses, which can differ for individual viral strains. The use of the optimal parameters of avian influenza virus replication in the MDCK cell lines yields virus titers comparable with virus reproduction in the chick embryos. The reproductive studies of the same avian influenza virus strains in chick embryos have shown that the maximum virus multiplication is seen when observing the optimum incubation time for infected embryos, which may be dissimilar in different strains. A considerable increase in hemagglutinin output can be achieved on adding trypsin to the infected chick embryos.

Alkaline versus neutral cultivation of bovine rotavirus strain 101 in the continuous cell cultures resulted in a significant acceleration of a reproduction cycle with a very significant cytopathic effect.

The epidemic upsurge of influenza morbidity in Russia in 2004-2005 was caused by the active circulation of influenza A(H3N2) and B viruses. A hundred and sixty-six epidemic strains were studied. All the strains were isolated in the MCK cell culture. Influenza A(H3N2) viruses (n=77) were antigenic variants of the reference A/Fujian/411/ 2002 and A/California/7/2004 strains. Three influenza A(H1N1) viral strains that were antigenic variants of A/New Caledonia/20/99 strains were isolated in sporadic cases. Influenza B virus strains (n=83) were antigenic variants of the reference B/Shanghai/361/02--lineage B/Yamagata/l6/88. In addition, 3 antigenic variants of B/Hong Kong/ 330/2002 (lineage B/Victoria/2/87) strains were isolated. Nine (20%) strains resistant to rimantadine at a concentration of 5 microg/ml were identified. Chromatographic analysis of B/Shanghai/361/02 and BIHong Kong/330/01 viral protein M1 trypsin hydrolysates revealed differences in the profiles of chromatograms of influenza A virus proteins M1. Examination of 121 pair sera from patients revealed an increase in antibodies to influenza A(H3N2) viruses in 10-21% of cases and to influenza B viruses in 20-36% of cases.

A collection of thermophilic strains of the genus Bacillus was made. The strains were screened for antimicrobial activity. Strains VK2 and VK21 isolated from thermal springs of the Kamchatka Peninsula, and antagonistic to several gram-positive bacterial species were chosen for further investigation of antibiotics produced by them. Restriction analysis of DNA coding for 16S rRNA showed that both strains can be assigned to Bacillus licheniformis. It was shown that the lytic activity of strains VK2 and VK21 was not related to the synthesis of hydrolytic enzymes. The maximum level of antimicrobial activity in the growth medium was found to correspond to the beginning of the stationary growth phase. Addition of manganese sulfate induced sporulation and altered significantly the time course of antibiotic production in both strains. Active metabolites were extracted with n-butanol. They survived boiling for 30 min and were resistant to trypsin and chymotrypsin but were partly hydrolyzed by pronase. They were stable at a pH range of 2.0-9.0.

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off of C-peptide from proinsulin results in the formation of ArgB31-ArgB32-insulin. The effect of temperature on the formation of de-ThrB30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain no N-terminal Met residue.

The conversion of porcine and human proinsulins to insulin by enzymatic hydrolysis induced by trypsin and carboxypeptidase B was studied. The conditions of reactions in the presence of trypsin (pure or immobilized in PAAG) and carboxypeptidase B were determined. The possibility of insulin production by the use of immobilized trypsin and carboxypeptidase B for practical purposes was shown.

Systemic enzymes (wobenzime and wobe-mugos) were approved, that had been prescribed as part of polychemotherapy to 24 patients with malignant lymphomas, who presented with large masses of lymphatic nodes poorly resorbable against the background of polychemotherapy and who were assigned for a high-total dose irradiation, which fact would undoubtedly entail sclerosing of adjacent intact tissues. The use of the enzymes made for the optimum realization of the polychemotherapy effect and permitted avoiding a good many of undesirable side events and complication thereof. The above systemic enzymes are well tolerated by patients; they induce no significant changes in the cellular composition or in blood biochemical indices.

The optimal conditions of enzymatic hydrolysis of proteins different composition and structure (protamin, casein, haemoglobin, fibrin, collagen) were established by using proteolytic enzymatic preparations with different specificity (pancreatin, pilrin, hepatopancreatin). The degree of hydrolysis, molecular and amino acid compositions of proteins were determined.

Method for direct isolation of heme peptide from enriched yeast biomass have been developed. This method makes it possible to eliminate the process of cytochrome c purification. Amino acid sequence for the Candida valida heme peptide is proposed. Exact molecular masses for both cytochrome c and its heme peptide are determined by mass-spectrometry.

Collase, an enzymatic preparation made from crab hepatopancreas, was used as a dissociative reagent in preparation of primary cell cultures and for detachment of continuous cells from the substrate during reinoculation. Collase was found to increase viable cell harvest by 2 to 2.5 times in comparison with trypsin and had a less detrimental effect on the cells which retained their proliferative activity, morphology, productivity, and sensitivity to viruses.

Prostaglandin H synthase (PGHS) as apo- and holoenzyme and the enzyme inactivated during the conversion of arachidonic acid into prostaglandin H2 has been modified by diethyl pyrocarbonate (DEPC). DEPC (40 mol/l mol protein) rapidly, but quantitatively differently interacted with the three forms of the enzyme (pH 6.0, 25 degrees C). The exhausted reaction with DEPC corresponded to modification of seven histidine residues in apo-PGHS and four residues in holo-PGHS. All of the 18 histidine residues were available for modification in the enzyme inactivated during the catalysis. The modification of apo-PGHS was accompanied by a concerted loss of the combined cyclooxygenase plus peroxidase and peroxidase activities. The velocities of the tryptic cleavage of the three forms of the enzyme into the 33 and 38 kDa polypeptides were essentially different, but the modification of each enzyme form did not affect the velocity of its cleavage. Two of the three histidine residues essential for the interaction with the heme within the 38 kDa fragment might be His-309 and His-388. Based on the comparison of availability for the reaction with DEPC of all the 18 histidine residues in the enzyme molecule inactivated by the interaction with arachidonic acid and on the abnormally high velocity of the tryptic cleavage of this form of PGHS, a hypothesis has been put forward about the fast and dramatic changes in the protein structure in the course of catalysis.

Solubilization of proteins from total membranes (a mixture of cytoplasmic and outer membranes) of Rickettsia prowazekii, a typical gram-negative bacterium, was studied using three different detergents. It was shown that isolated outer membranes and sarkosyl-insoluble material contain major polypeptides of 134, 31, 29.5 and 25 kDa as well as minor polypeptides of 78, 60, 42, and 17 kDa, while the total membranes--the same plus a great number of additional minor proteins. The material solubilized by octyl glucoside in the presence of MgCl2 contains exclusively major proteins (134, 31, 29.5, and 25 kDa). No differential solubilization takes place upon membrane treatment with octyl glucoside in the absence of Mg2+ or with Triton X-100. Rickettsial proteins are insensitive to trypsin in both whole cells and total membranes, unless the latter are presolubilized with octyl glucoside. Proteinase K degrades all of the total membrane proteins but only the 134 kDa polypeptide of whole cells. Upon immunoblotting predominantly the major outer membrane proteins (134, 31, and 20.5 kDa) and, to a lesser extent, the minor proteins (60, 42, and 17 kDa) interact with human convalescent serum.

Histones' H2A and H2B peptidic points which interact with nucleosomal DNA have been identified by using the methods of DNA--protein covalent cross-linking. H2B can be linked to DNA via its N-terminal tail and via several lysines contained within residues 24-34. The most prominent site of histone H2A covalent linking to DNA is His-123, the less prominent being Lys-119 and Lys-124.

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.