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Klebsiella pneumoniae is a nosocomial pathogen that exhibits multidrug resistance and virulence. Emergence of carbapenem-resistant K. pneumoniae is increasingly reported worldwide, especially strains producing OXA-48 carbapenemases, Ambler class D beta-lactamases encoded by blaOXA-48 gene. These strains cause difficulties to detect in clinical specimens due to many of these isolates falling within the susceptible range for carbapenems. The aim of this study was to identify specific characteristics of K. pneumoniae isolates containing OXA-48 gene. Four clinical isolates were chosen for the analysis, two containing OXA- 48 gene and two strains lacking OXA-48 gene (controls). OXA-48 genes presence was confirmed by a multiplex polymerase chain reaction (PCR). The antimicrobial susceptibility was examined by disc diffusion method and by determination of minimal inhibitory concentrations (MIC) with broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. Two isolates with present OXA- 48 gene were susceptible to carbapenems (imipenem and meropenem). To distinguish the difference between the analysed strains, a comprehensive proteomics analysis was performed via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) operating in data‐independent acquisition mode (DIA). DIA technology enabled accurate identification of more than 2000 proteins in all of the strains. Beta- lactamase OXA-48 was detected in OXA-48 positive strains despite low- level resistance. Quantitative analysis revealed an altered expression of membrane proteins in OXA- 48 strains in comparison to controls. Particularly, upregulation of putative porin KPN_04773 and outer membrane lipoprotein SlyB were detected in OXA-48 strains. Furthermore, outer membrane protein OmpF was downregulated in OXA-48 strains. As well, downregulation of peptidase YggG and two proteins with dehydrogenase activity (KPN_01492 and PutA) was also detected in OXA-48 strains. Overall, our results give an insight into the difference in the protein expression between OXA-48 strains and controls and indicate that OXA- 48 gene presence is not the only difference between strains.

Osnovni cilj ovog istraživanja bio je utvrditi promjene kemijskih parametara svježine (histamina, trimetilamina i dimetilamina) lubina Dicentrarchus labrax tijekom skladištenja na različitim temperaturama (4±1°C i 12 ±1°C ). Pored toga, uspoređena je učinkovitost polusatnog tretmana ribe kupkom s 0, 9% NaCl u odnosu na klasično pakiranje ribe u podloške direktno s leda. Također je uspoređen i učinak dvije najčešće korištene vrste stiropornih podložaka na kemijske pokazatelje svježine ribe.

Western blotting of (a) p53, (b) hsp70 and (c) TNF proteins detected in normal fibroblast HS-68 cell lines after 48 h exposure to a 0.1% concentration of Anisakis excretory/secretory (ES) and crude extract (EC) products: Lane 1: control; Lane 2: ES exposure; Lane 3: EC exposure, Std, mix of standard proteins as molecular markers. *P

Humans can become accidental host after consumption of raw or thermally inadequately treated fishery products that are contaminated by alive third-stage (L3) Anisakis larvae. Different types of anisakiasis have been recognised ; gastric, intestinal, ectopic or gastroallergic anisakiasis, in hypersensitized patients. In countries where both the prevalence of human cases and general awareness of this zoonosis is high, there have been described rare cases of bowel carcinomas associated to the isolated larvae. It is still unclear if two aetiologies, e.g. anisakiasis and carcinoma are related or accidental incidences, but it is indicative that in all reported cases, neoplasia and embedded larvae shared the common site affected by chronic inflammation. In line with this, the aim of our study was to test the effect of A. pegreffii total antigen (ATA) on human normal fibroblast cell lines HS-68 and subsequently measure the level of oxidative stress (ROS), inflammatory and carcinogenic markers (Hsp70, p53, c-fos, c- jun and TNF). Two types of ATA were used ; the first obtained from parasite excretory/ secretory production (ES), and the second obtained by whole-nematode ethanol extraction (CE). Preliminary results evidenced that cells have undergone a progressive reduction in vitality after ATA stimulation in dose and time dependent matter, but the effect differed in respect to the antigen product applied. Similarly, ROS was significantly increased compared to control cell line, and highly correlated to cell vitality. Expression of p53, c-foc and c-jun, quantified by q-PCR was congruent to immunoblotting detection of Hsp70 and TNF at protein level, all being significantly upregulated in affected cells. This suggests that Anisakis total antigen products can indeed trigger two pathways ; the first one is oxidative stress pathway characterized by inflammatory and apoptotic pattern in cells subjected to Anisakis excretory/ secretory products, while the other is more toxic effect characterized by induction of carcinogenic markers and blockage of inflammation and apoptosis in cells subjected to parasite crude extract.

Nonribosomal peptide synthetases (NRPS) are large multifunctional enzymes that catalyse the synthesis of nonribosomal peptides (NRP) known for a broad range of pharmaceutical properties such as antimicrobial, immunosuppressive and antitumor activity. With overwhelming reports of bacterial resistance, it is of fundamental importance to gain more insight into structural and functional properties of these megastructures. Biosynthesis of NRP starts with the adenylation (A) domain that activates a specific amino acid substrate in form of aminoacyl-adenylate with concomitant release of pyrophosphate. The activated amino acid is subsequently transferred via the peptidyl carrier protein (PCP) domain to the neighbouring active site of the condensation (C) domain that finally catalyses peptide bond formation. Crystallographic studies on various NRPS A-domains showed that these enzymes undergo extensive structural rearrangements during the catalytic cycle. Two catalytically distinct conformations are reported for A-domains: the first being implied in adenylate formation, and the second in transfer of the activated amino acid onto the PCP-domain. Here we report the first extensive study on fluorescence properties of a representative A-domain from tyrocidine synthetase 1 (TycA-A). TycA-A comprises five potentially fluorescent Trp residues designated W227, W301, W323, W376 and W406, respectively. Individual Trp accessibility surface area (ASA) and their structural position were assessed based on a structural model of the TycA-A protein in both conformations. To resolve which Trp contributes most to the emission spectrum of TycA-A, single point mutants bearing Trp to Phe substitutions were constructed. Mutant proteins were tested for thermal and structural stability using the thermal shift assay and limited proteolysis. Acrylamide quenching was employed to probe accessibility of individual Trp residues upon substrate binding to mutant and wild type protein, respectively. Our results show that among five Trp residues only W227 reports to substrate binding. Conformational changes upon non-cognate amino acid binding was also probed by acrylamide quenching in a mutant protein bearing only W227. This showed that the extent of quenching is more evident with non-cognate substrates, indicating a suboptimal overall conformation with concomitant hydrolysis of the aminoacyl-adenenylate.

Analysis of phycotoxins in tissue of the shellfish Mytilus galloprovincialis (Lamarck, 1819) have been carried out during the summer period of 2007 in the area of the Southern Adriatic, where no similar studies were done before. Mussel samples were collected from a localized site in Dubrovnik area during summer 2007 from July to September, twice a month. At the same time, sea-water samples were collected in order to find the presence of toxic phytoplankon species. Analysis of shellfish toxicity was performed by mouse bioassay, HPLC, and LC-MS/MS. YTX and its analogue 45-OH YTX were detected. Gymnodimine was also measured, for the first time in Adriatic Sea. Phytoplankton analysis of seawater samples, collected in the investigated area, showed the occurrence of potential sources of YTX: Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. Potential source of Gymnodimine, Gymnodinium, was also detected.

Nonribosomal peptide synthetases (NRPSs) are modular proteins responsible for the production of peptide natural products, many of which have antibiotic activity. The modular nature of NRPS makes them suitable targets for engineering through domain or module rearrangements. In practice, the ability to obtain functional enzymes depends on the extent to which the protein-protein interactions necessary for catalytic activity of the engineered NRPS assembly have been preserved. High-resolution NMR and crystal structures of NRPS domains, didomains and even an entire module obtained during last several years greatly expanded our understanding of intra- and intermodular protein interactions in NRPS systems. Distinct protein-interaction surfaces were reported mainly for the peptidyl carrier protein (PCP) domain ; however, several attempts to map protein interaction surfaces on adenylation (A) domain failed to pinpoint a particular region of the protein. Our search for the putative surface on the A domain, involved in protein interaction with the PCP domain was based on sequence analysis and homology modeling. We examined all the available structural data not only from NRPS A domains, but also of the other members of adenylate-forming superfamily, and hypothesized that the region of A domain assigned as A9 core motif could be of importance for A and PCP domain interaction. When analyzing homology models build for the protein conformation in the first half-reaction (amino acid activation) and during the second half-reaction (transfer of the activated amino acid to the 4’-phosphopantetheine arm of the adjacent PCP domain), this region seems to position itself favorably for the interaction with PCP domain during the second half-reaction. To test this hypothesis, we created a set of mutant proteins, in which single amino acid exchanges were introduced into the A9 core motif of A domain from tyrocidine synthetase 1, 484LPAYMLP. The mutant proteins were expressed and purified from E. coli as His-tagged proteins. The influence of mutation in A9 motif was examined by assaying enzymatic activity (with or without the acceptor of activated amino acid in the second-half reaction), susceptibility to proteolysis, and fluorescence properties of the mutant enzymes. The results obtained indicate that, while the A9 core motif is not indispensable for the activation of amino acid, it contributes to the structural stabilization of the protein and might play an important role for the transfer of activated amino acid from the adenylation onto the PCP domain.

Istraživan je biokemijski sastav amfipodnog račića Gammarus pulex (L.1758) sakupljenog u svibnju 2009. godine na izvoru rijeke Lađenice (Glavatičevo-Konjic). Izmjerena je ukupna koncentracija bjelančevina, lipida i ugljikohidrata kako bi se utvrdila nutritivna vrijednost račića. Dobiveni rezultat ukazuje na vrlo visok udio bjelančevina u uzorku. Ukupni udio bjelančevina (65%), kao i udjeli lipida (6, 5%) i ugljikohidrata (2, 5%) svrstavaju ovaj organizam u red najkvalitetnije žive hrane i opravdavaju daljnja istraživanja u svrhu mogućnosti uvođenja gamarida u komercijalni uzgoj slatkovodnih, boćatih i morskih organizama.

Nonribosomal peptide synthetases (NRPS) are modular proteins that catalyze the synthesis of small peptides with antibiotic, immunosuppressant, and anticancer activities, as well as siderophores. NRPS usually contain one module for each amino acid incorporated into the final peptide. Each module consists of several catalytic domains that catalyze the activation of specific amino acids (adenylation (A) domain), covalent thioester binding (peptidyl-carrier-protein (PCP) domain), formation of peptide bond (condensation (C) domain), and optionally, various substrate modifications. A domain catalyzes the two-step reaction of ATP-driven activation of amino acid, followed by its transfer to PCP domain. Sequence alignments of A domains allowed identification of 10 ‘ core motifs’ . Most of them were assigned particular functions, thanks to crystal structures and mutagenesis. Unequivocal function for several conserved motifs has not been established so far. Recently, a significant progress in mapping protein interactions between individual NRPS domains has been achieved. However, regions important for A and PCP domain interaction were not identified. In our previous work we were studying the fidelity of substrate selection by A domain, using tyrocidine synthetase from B. brevis as a model system. Our present work is focused on investigating the role of conserved sequence motifs, especially those that are shown to adopt strikingly different conformations during the two-step catalytic reaction. We are also interested in examining A domain for putative protein-protein interaction surfaces. We hope that the result obtained in these studies will facilitate the rational design of NRPSs as a means of producing peptides with novel biological activities.

This paper reports the first occurrence of yessotoxin (YTX) in the shellfish Mytilus galloprovincialis (Lamarck, 1819), collected from a localized site in the Trogir bay, in the costal area of the eastern-mid Adriatic Sea (Croatia). Mussel samples were collected in the summer of 2006 from July to October, twice a month. Analysis of shellfish toxicity was performed by mouse bioassay, HPLC and ELISA. The obtained results demonstrate the predominant presence of YTX. Phytoplankton analysis of sea water samples, collected in the investigated area, showed the occurrence of two potential sources of YTX, Lingulodinium polyedrum and Gonyaulax spinifera.

Alkalna fosfataza (AP, E.C. 3.1.3.1) je nespecifična fosfomonoesteraza koja katalizira hidrolizu velikog broja fosfomonoestera. AP pokazuje odstupanja od Michaleis-Menten kinetike koja se mogu objasniti modelom dimernog enzima s nejednakim podjedinicama. Predloženi model opisuje mehanizam hidrolize supstrata uslijed konformacijskih promjena na dodirnoj površini podjedinica. Značaj dodirne površine podjedinica i β -nabrane ploče koja se proteže ispod aktivnog mjesta do površine podjedinice, ispitana je metodom usmjerene mutageneze kojom su dobiveni mutantni enzimi s jednom (T81A), odnosno dvije (T81A/Q83L) mutacije. Mutirane aminokiseline Thr-81 i Gln-83 smještene su u β -nabranoj ploči koja se nalazi na dodirnoj površini između susjednih podjedinica. Mutirani enzimi su pročišćeni i uspoređeni sa divljim tipom AP. Analizirani su kinetički parametari te su ispitana dinamička svojstva enzima mjerenjem fosforescencije pri sobnoj temperaturi. Kinetičke karakteristike ispitivane su u 1 M Tris/HCl, pH 8, te u 0, 35 M 2A2M1P, pH 10, 5 puferu. Mutacije na dodirnoj površini podjedinica, utjecale su na strukturnu stabilnost proteina i na njegove kinetičke karakteristike, ukazujući na važnost interakcije dodirnih površina na katalitički mehanizam.

Yessotoxin (YTX) and its derivates are lipophylic disulphated polyeter algal toxins structurally related to ciguatoxins and brevetoxins. It was originally isolated from scallops Patinopecten yessoensis in Japan. Also, it has been found in shellfish from Norway, Chile, Italy, New Zealand and along the Russian coast of White Sea. YTX was also established as a causative agent of toxicity detected by mouse bioassay in the Northern Adriatic Sea. Its presence in the Northern Adriatic has been confirmed by liquid chromatography-mass spectrometry (LC-MS). The present study investigated the occurrence of YTX in the shellfish Mytillus galoprovincialis, collected in the summer of 2006, from July to October, from a localized site in Trogir Bay ; and from July to September from the Dubrovnik City Port. Analysis of shellfish toxicity was performed by LC-MS and enzyme-linked immunosorbent assay (ELISA). Analysis was supplemented by mousse bioassay. Along with shellfish toxicity, phytoplankton composition was investigated. The presence of YTX was established in several samples from Trogir and Dubrovnik. Phytoplankton analysis of sea water samples, collected in the investigated area, showed the occurrence of known YTX producers Protoceratium reticulatum, Lingulodinium polyedrum and Goniaulax spinifera.

Alkaline phosphatase (APase, E.C. 3.1.3.1) is a non-specific phosphomonoesterase catalysing the hydrolysis of a large number of phosphomonoesters. Most unresolved questions, relating to the catalytic mechanism of APase concern the influence of conformational changes and allosteric interactions on catalytic efficiency. APase displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. The importance of the subunit interface and the β -pleated sheet stretching from underneath an active site to the subunit surface in the transfer of conformational information, has been assessed by site-directed mutagenesis followed by kinetic analysis and probing of dynamic properties of the APase dimer using room temperature phosphorescence. The mutant APase, carrying alanine in place of Thr81, as well as a double mutant carrying alanine in place of Thr81 and leucine in place of Gln83 were analyzed in comparison to the wild type protein. Amino acid residues Thr-81 and Gln-83 are located within the β -pleated sheet at the contact surface between the subunits, and form hydrogen bonds with analogous amino acids from the adjacent subunit. The mutant proteins were purified and compared with wild type APase. Kinetic properties have been determined in 1 M Tris/HCl, pH 8, and in 0.35 M 2A2M1P, pH 10.5. Mutations introduced at the subunit interface affected both structural stability of the protein and its kinetic properties emphasising the importance of subunit interaction in the catalytic mechanism.

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non- equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [Orhanović S., Pavela-Vrančič M. (2003) Eur. J. Biochem. 270, 4356-4364]. The significance of interactions at the subunit interface and the involvement of the β -pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild type protein. The T81A mutant proved to be more sensitive to thermal denaturation, and undergoes irreversible dissociation to monomers upon depletion of metal ions by EDTA. The affinity of T81A for the substrate and Pi was slightly reduced in 2A2M1P buffer at pH 10.5. In 1 M Tris/HCl, pH 8, the turnover number kcat of the T81A mutant was reduced compared to the wild type enzyme, indicating that the mutation, affecting interactions between β -pleated sheets at the subunit interface, influences the rate determining step at pH 8. The T81→ A mutation, introduced at the subunit interface, affected both structural stability of the protein and its kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. Rigid parts in the polypeptide structure supposedly accomplish the transfer of conformational information between the active sites across the subunit interface. The importance of the subunit interface and the β -pleated sheet, stretching from underneath an active site to the subunit surface, in the catalytic mechanism has been probed by site-directed mutagenesis. The mutation replacing Thr-81 with alanine and Gln-83 with leucine were introduced into the APase gene. Amino acid residues Thr-81 and Gln-83 are located within the β -pleated sheet at the contact surface between the subunits, and form hydrogen bonds with analogous amino acids from the adjacent subunit. Two kinds of mutant proteins were prepared, a single T81A and double T81A/Q83L mutant. The mutant proteins were purified and compared with wild type APase. Stability of the protein was assessed by thermal denaturation. The affinity for metal ligands and their influence on the quaternary structure was examined by monitoring enzyme inactivation in the presence of EDTA. Kinetic properties have been determined in 1 M Tris/HCl, pH 8, and in 0.35 M 2A2M1P, pH 10.5. Mutations introduced at the subunit interface affected both structural stability of the protein and its kinetic properties emphasising the importance of subunit interaction in the catalytic mechanism.

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. Rigid parts in the polypeptide structure supposedly accomplish transfer of conformational information between the active sites across the subunit interface. The importance of the subunit interface and the β -pleated sheet, stretching from underneath an active site to the subunit surface, in the catalytic mechanism has been probed by site-directed mutagenesis. The mutation replacing Thr-81 with Ala was introduced into the APase gene. Thr-81 is located within the β -pleated sheet at the contact surface between the subunits, and forms hydrogen bonds with the analogous Thr-81 from the adjacent subunit. The mutant protein was purified and compared with the wild type APase. Stability of the protein was assessed by thermal and urea denaturation. The affinity for metal ligands and their influence on the quaternary structure was examined by monitoring the inactivation of the enzyme in the presence of EDTA. Kinetic properties have been determined in 1 M Tris/HCl, pH 8 and in 0.35 M 2A2M1P, pH 10.5. The mutant enzyme proved to be more sensitive to inactivation with EDTA and to thermal denaturation. Following dialysis in the presence of EDTA the mutant enzyme dissociated into subunits contrary to the wild-type protein that maintains its dimeric structure. The activity of the mutant protein could not be reconstituted upon addition of metal ions. The affinity of the mutant enzyme for the substrate and for inorganic phosphate was reduced in 2A2M1P buffer at pH 10.5. The mutation introduced at the subunit interface affected both structural stability of the protein and its kinetic properties emphasising the importance of subunit interaction in the catalytic mechanism.

Introduction: A variety of bioactive natural peptide products is produced by several bacterial and fungal species via the protein thiotemplate mechanism catalysed by non-ribosomal peptide synthetases (NRPS). NRPSs are composed of modular proteins, each module consisting of a set of apparently independent structural and functional domains that bind, activate and condense the carboxyl substrate into the growing polypeptide chain. Although a large body of information is available on the structure-function relationship of peptide synthetases, much of the basic aspects concerning the molecular dynamics in maintaining amino acid recognition specificity and regulation of the biosynthetic process still remain unclear. In this work, analysis of the anticipated ATPase activity of NRPS was examined. To assess whether this activity is relevant in the presence of the amino acid substrate, the reaction was followed in the presence of a number of naturally occurring amino acids and amino acid analogues known to support variable degrees of adenylation efficiency. Materials and Methods: Apo-tyrocidine synthetase 1 (apo-TY1), gramicidin S synthetase 2 (GS2) and δ -(L-α -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), were purified from recombinant E. coli, Bacillus brevis ATCC9999 and Acremonium chrysogeneum, respectively. A coupled spectrophotometric assay involving purine nucleoside phosphorylase assay was applied to follow the continuous turnover of Pi in the presence of NRPS, ATP and amino acid. Results: Besides ATP-dependent adenylation activity, here we show that multifunctional NRPS demonstrate an ATPase activity, in the range of up to 2 Pi/min. Whereas the cognate amino acids display, in general, a negative effect on the ATPase activity, the presence of non-cognate substrates increases the rate of Pi generation. The effect is less pronounced with ACSV, which is a complete biosynthetic system, than with apo-TY1 (monomodular component of the tyrocidine biosynthetic system catalysing the activation of a single amino acid, L-Phe) or GS2 (tetramodular component catalysing the activation of L-Orn, L-Pro, L-Leu and L-Val). The enhanced hydrolysis rate by apo-TY1 in the presence of non-cognate amino acid substrates correlates well with their structural features and the diminishing adenylation efficiency. Conclusion: The adenylation domain of the multifunctional NRPSs demonstrates, besides ATP-dependent adenylation activity, an additional property related to ATP binding and hydrolysis, which might be part of the substrate recognition mechanism. This knowledge may prove useful in exploring the regulatory mechanism of peptide synthetases, relevant in exploiting their combinatorial potential for the generation of novel and more effective variants of existing drugs, or using the non-ribosomal chemistry for the preprogrammed generation of new peptide-forming systems.

Katalitička svojstva ALP studirana su u ovisnosti o pH, Zn2+, Mg2+, anorganskog fosfata te o inkubacijskom periodu. Mehanizam aktivacije uključuju unterakcije među podjedinicama. Asimetrija dimernog enzima olakšava otpuštanje fosfata konformacijskom promjenom koja smanjuje afinitet prema supstratu i produktu.

Alkaline phosphatases (AP) are nonspecific phosphomonoester hydrolases. They are found in most species from bacteria to man indicating an involvement in fundamental biological processes. All APs are mulitmeric metalloproteins found outside the cell, in mammals attached to the membrane via a phosphatidyl inositol anchor or residing in the periplasmic space of bacteria. Kinetic and structural properties of AP have been thoroughly investigated using various spectroscopic and kinetic techniques. AP displays a characteristic kinetic behaviour, commonly described as the negative cooperativity or the half-of-the-sites reactivity. Most unresolved questions are related to conformational changes in the catalytic cycle, allosteric interactions and their influence on the catalytic efficiency. AP from E. coli is often used as a model enzyme system to study the role of metal ions in catalysis, and for the investigations of the characteristic properties of oligomeric enzymes such as negative cooperativity or the half-of-the-sites reactivity, and interactions between subunits. In addition, it could be used as a model to investigate the potential advantage of dimeric enzyme, with such kinetic properties, over monomeric enzymes. Our study is focused on understanding the role and nature of subunit interactions in the catalytic mechanism of AP from E. coli. To investigate the role of specific amino acid in establishing communication between the monomers, site-directed mutagenesis on the E. coli alkaline phosphatase gene (phoA) has been conducted. PhoA was amplified from genomic DNA using the polymerase chain reaction, and inserted into a pET expression vector. Overproduction of wild-type PhoA was confirmed by SDS-PAGE and the activity assay. The mutant enzyme was created by replacement of Thr81, located at the subunit interface and hydrogen-bonded to its counterpart of the other subunit, with alanine.

Alkaline phosphatase (ALP) is a non-specific phosphomonoesterase catalysing the hydrolysis of a large number of phosphomonoesters. Although the exact biological role of ALP is not yet known, its presence in almost all forms of life implies its participation in fundamental biological processes, most likely in phosphate transport. ALP from E. coli is a dimeric metalloprotein with three metal binding sites per active centre. The crystal structure has been determined revealing direct participation of two zinc ions in substrate binding and phoshomonoester hydrolysis, while the role of Mg2+ in the kinetic mechanism has remained unexplained. Although ALP crystalises as a symmetric dimer, it displays deviation from Michaelis-Menten kinetics, supported by a model of a dimeric enzyme with unequal subunits. It has been determined that ALP contains only 1.3 ± 0.2 moles of Mg2+ per dimer indicating that negative cooperativity in Mg2+ binding could be an explanation for the observed asymmetry. The influence of the metal ion content on the catalytic properties of ALP from E.coli has been examined by kinetic analysis. Alterations in tertiary structure in dependence of the metal ion concentration were tested using limited proteolysis with trypsin. An increase in ALP activity due to catalytically relevant metal ion binding was accompanied with structural changes resulting in reduced susceptibility to proteolysis. The reduction of ALP activity in the presence of higher metal ion concentrations is probably the consequence of complete saturation of the dimer with metal ions.

Alkaline phosphatase is an extracellular enzyme enabling the utilisation of phosphomonoesters as a source of Pi required for the maintenance of the cellular metabolism. Its catalytic properties have been extensively studied in aquatic environments in an attempt to describe its influence on phosphorous cycling in natural waters. Despite its significance and frequent determination a standard method has not been developed yet. In order to examine the effect of various reaction conditions on the kinetic properties of ALP in seawater the stability of ALP in chloroform-stabilised samples has been studied in addition to the influence of the buffer type, its concentration and pH, the metal-ion content, Pi and incubation time. It could be concluded that the kinetic properties of ALP are significantly influenced by the buffer pH, the concentration of Mg2+ and Zn2+ ions and the incubation period, requiring a precise definition of these reaction parameters in determining a standard method for measurement of ALP activity in water media.

Alkaline phosphatase is a multimeric metalloprotein catalyzing phosphohydrolytic and phosphoryl transfer reactions. It is present in the periplasmatic space of prokaryotes or associated with the outer side of the plasmatic membrane in eukaryotes, suggesting its involvement in phosphate transport into the cell. According to the crystallographic study, alkaline phosphatase from E. coli (PhoA) is composed of two apparently identical subunits. Three classes of metal binding sites exist in active site on each subunit, two of which bind Zn and one Mg ion. A vast amount of data exists indicating a non-equivalence of the PhoA subunits, including Pi binding with strong negative cooperativity, biphasic thermal inactivation and deviations from Michaelis-Menten kinetics substantiated by enzyme asymmetry. Unequal saturation of the subunits with metal ions could induce the observed enzyme asymmetry. We have used limited proteolysis as a probe of conformation to examine the possible structural differences between the subunits of alkaline phosphatase. Susceptibility of the enzyme to digestion with proteinase K or trypsin was studied in solutions containing different concentrations of Zn and Mg. The progress of the proteolytic reaction was monitored by PhoA activity assay and SDS-PAGE. The proteolytic pattern and the time course of digestion were dependent on the concentration of metal ions in the reaction solution. Metal-free alkaline phosphatase was highly susceptible to proteolysis, while the presence of bound metals provided protection form digestion under the same reaction conditions. We conclude that binding of metal ions to alkaline phosphatase induces conformational changes in the protein that alter its susceptibility to proteolysis and influence its enzymatic activity.

Alkaline phosphatase (PhoA) is a multimeric metalloprotein catalyzing phosphohydrolytic and phosphoryl transfer reactions. It is present in the periplasmatic space of prokaryotes or associated with the outer side of the plasmatic membrane in eukaryotes, suggesting its involvement in phosphate transport as a possible biological role of alkaline phosphatases. According to the crystallographic study, PhoA from E. coli is composed of two apparently identical subunits. On the other hand, a vast amount of data exists indicating a non-equivalence of the PhoA subunits, including Pi binding with strong negative cooperativity, unequal saturation with Mg2+, biphasic thermal inactivation and deviations from Michaelis-Menten kinetics substantiated by enzyme asymmetry. These data suggest conformational heterogeneity between the subunits. It has been reported that PhoA from E. coli is not fully saturated with metal ions. Unequal saturation of the subunits could induce the observed enzyme asymmetry. The dependence of the kinetic constants and the deviations from Michaelis-Menten kinetics on the Mg2+-ion concentration has been investigated. We have examined the possible structural differences between the subunits using limited proteolysis with trypsin as a probe of conformational changes. Susceptibility to proteolysis was studied in solutions containing different concentrations of metal ions and a metal-complexing agent. A variable kinetic behaviour of PhoA was observed. The progress of the proteolytic reaction was monitored by SDS-PAGE and the PhoA activity assay. The proteolytic pattern and the time course of tryptric digestion were dependent on the concentration of metal ions in the reaction solution. We conclude that binding of metal ions to PhoA induces conformational changes in the protein that alter its susceptibility to proteolysis and influence its enzymatic activity.

Peptide synthetases constitute a family of multifunctional enzyme systems involved in the non-ribosomal biosynthesis of a variety of microbial peptide products, some with important medical and biotechnological applications as antibiotics (gramicidin S, tyrocidine, penicillins), immunomodulators (cyclosporin), extracellular surfactans (surfactin), and sideophores (enterobactin). The modular construction of the polypeptide chain opens the possibility for manipulation, and consequently generation of novel peptides with modified biological activity. Substrate activation is accomplished by formation of an acyladenylate, followed by charging of the activated moiety onto the sulfhydryl group of the 4˘- phosphopantetheine cofactor for subsequent modification and elongation. The crystal structure has provided a wealth of structural information, however the substrate binding sites, and the conformational changes which occur upon binding and prior to product release are currently unresolved issues. The major unanswered questions about the dynamic behaviour of peptide synthetases regard the binding of ATP, the juxtaposition of the b- and g-phosphates for adenylate formation, the mechanism of PPi release, and the significance of the thioesterification site in regulating the conformational stability of the activated intermediates. The functional importance of the two flexible loops comprising the most conserved residues, Arg416 and Lys186, has been studied by the use of mutant analysis, limited proteolysis, and chemical modification of tyrocidine synthetase 1. It has been proposed that the (S, T)GT(T, S)GxPKG signature sequence is involved in orienting the b- and g- phosphates for the catalytic reaction. The 4˘- phosphopantetheine cofactor is likely to be relevant for maintainance of the active site conformation, and consequently protection from decomposition by hydrolysis.

Alkaline phosphatase is nonspecific fosfomonoesterase catalysing phosphohydrolytic and phosphoryl-transfer reactions. Biological function of ALP is still unclear. The widespread occurence of ALP in nature implicates involvment in fundamental biological processes, most likely in phosphate transport. Alp is multimeric metalloprotein with three metal ion binding sites pewr active site. Two zinc ions directly participate in substrate binding and catalysis while the role of the magnesium ion is not fully understod yet. Alp shows deviations from Michaelis-Menten kinetics what could be substantiated by a model assuming nonequivalent subunits. It has been reported that Alp from E.coli is not fully saturated with magnesium ions. Unequal saturation of the subunits could induce observed enzyme asymmetry. The dependence of the kinetic constants and deviations from Michaelis-Menten kinetics on the magnesium concentration has been investigated. The difference in subunit affinity for ligands as well as deviations from linearity are reduced in the presence of higher Mg 2+ concentration, as could be expected upon binding of Mg 2+ to both subunits. It has been established by limited proteolysis that difference in tertiary structure exists between enzymes of variable saturation with metal ions. Obtained results could provide insight in the role of ALP in the phosphate transport.

Phosphate is often the growth limiting factor in natural waters. Abiotic decomposition of organic phosphorous compounds is very slow under environmental conditions. Organic phosphorus compounds in water, either of biological origin or as products of human activities, can not be directly assimilated by planktonic organisms. Inorganic phosphate becomes available only upon enzymatic hydrolysis outside the cell. This reaction represents the key stage in phosphorus cycling in natural waters. It has been established that a significant number of organophosphorus compounds can be hydrolyzed by alkaline phosphatase (E.C. 3.1.3.1. ALP), an enzyme present at the outer side of the cell membrane. Natural substrates for ALP are unknown, as well as their affinity or rate of hydrolysis in vivo. The influence of the organic and inorganic phosphate concentration on species selection, and the mechanism of phosphate transport into the cell remain unresolved questions. The aim of this study was to improve the method for the determination of ALP activity in seawater samples. Since the synthesis of ALP depends on the phosphate concentration, the measured activity has been used as an indicator of phosphorus deficiency in a phytoplankton community. For that purpose the concentration of inorganic phosphate has been determined, simultaneously with the phytoplankton analysis of the same seawater samples.

In response to favorable changes in their environment, certain algal species undergo a rapid population increase and may form visible patches on the surface reffered as the "red tide". These blooms may be either toxic or noxious, that is either producing specific toxins or causing anoxia through the decay process. Exeptional blooms of toxic phytoplankton species and their association with shellfish toxicity as the result of toxin accumulation in shellfish meat has been known for long time. Due to process of strong urbanization of coastal areas, inadeqate industrialization, tourism, intensive aqaculture and other anthropogenic influences provoking severe eutrophication, there is an increasing frequency, intensity, duration, and geographic spreading of outbreaks. Concern about toxic algal blooms has arisen in recent years becouse of their impact on public health, and on the economics of shellfish aqaculture. Poisoning by metabolites from marine algae has been classified into paralytic (PSP), neurotoxic (NSP) amnesic (ASP), and diarrheic (DSP) shellfish poisoning. The most important compounds of the DSP complex are okadaic acid (OA) and an increasing number of its derivatives named dinophysis toxins (DTX). Threath to public health has been aggravated by a recent discovey that OA is a highly selective inhibitor of protein phosphatases and a potent tumor promoting agent. Since phytoplankton toxins represents a serious threat to human health, as well to fish and shellfish farming, monitoring programs have been established in many countries. Investigation of the toxin structure, their biological function, and the biosynthetic pathway are also currently in progress. Kaštela bay is semienclosed bay, heavily polluted by land sources, both of domestic and industrial origin. Red tide events have been observed there regulary every summer since 1980, when it first occured in the Vranjic basin, the most threatened part of the Bay. Despite the presence of toxic algae (Gonyaulax polyedra and Alexandrium minutum) no toxic blooms has been recorded up to 1993, when the mouse bioassay analysis showed the presence of DSP toxins, thought in low concentration. Investigations have been carried out every summer since 1993, using the biological (mouse bioassay) and chemical (HPLC) methods. Preliminary studies in the Central Adriatic confirmed the presence of toxin producing species, thus implying the necessity of further investigations of the still unresolved questions. Due to lack of adequate equipment, we are not in the position to perform all necessary investigations, however we wish to highlight the existence of growing problem concerning the potential development of shellfish toxicity.

Organski spojevi fosfora u vodi bilo da su biološkog porijekla ili da su nastali kao rezultat ljudske aktivnosti, ne mogu direktno biti asimilirani od strane planktonskih organizama. Anorganski fosfor koji je dostupan planktonskim organizmima nastaje enzimskom hidrolizom izvan stanice. Ova reakcija predstavlja ključni stadij u kruženju fosfora u vodi. Utvrđen je znatan broj organofosfornih spojeva koji mogu biti hidrolizirani uz pomoć alkalne fosfataze, enzima koji je prisutan izvan stanične membrane. Cilj ovog rada je poboljšati metodu za determinaciju aktivnosti alkalne fosfataze u morskoj vodi.

Although monitoring of toxic algae in the middle Adriatic has been conducted for many years, the analysis of shellfish toxicity by mouse bioassay and HPLC was initiated in the fall of 1994. The results of the HPLC analysis indicated the presence of DSP and PSP toxins in the shellfish from the Kaštela bay, although the concentrations were too low for an identification by mouse bioassay. In the summer of 1995 and 1996 DSP toxicity was confirmed by mouse bioassay and HPLC analysis. The presence of DSP toxins in shellfish has been associated with the occurrence of the Dinophysis species (D. sacculus, D. fortii, D. caudata, D. rotundata). PSP toxins, found only in traces, were associated with the occurrence of the Alexandrium minutum bloom. This study was directed to the detailed analysis of the species D. sacculus, an autochtonous species found in the Bay. Its presence in the phytoplankton community was recorded from May to October, simultaneously with the occurrence of shellfish toxicity.

Although harmful algal blooms (red tide, "mucillagine") have been present in the Adriatic Sea for almost thirty years, toxic blooms are a relatively new occurrence in this area. This phenomenon was first recorded in 1989 off the northwest coast of the North Adriatic, the dinoflagellate of ginus Dinophysis being the causative organism of diarrhetic shellfish poisoning (DSP). In this paper we present the first data substantiating the occurrence of Diarrhetic Shellfish Poisoning (DSP) in the central region of the Adriatic Sea (Kaštela bay), registered in the summer of 1995. Toxicity was established using the mouse bio-assay, however the results of the HPLC-directed analysis suggest the observed toxic effects are not associated with the common DSP toxins. The HPLC analysis has shown the presence of an unknown compound structurally related to DTX-2, the concentration of the respective substance being rather high in those samples exhibiting toxicity. In a simultaneously conducted analysis of the phytoplankton population special attention was dedicated to the species from the dinoflagellate group. The origin of mussel toxicity can be traced to Dinophysis sacculus, a suspect toxic species.

A wide range of phytoplankton species has been found to produce a variety of toxic metabolites responsible for shellfish poisoning, and consequently human intoxication. Poisoning by metabolites from marine alga has been classified into paralytic (PSP), diarrheic (DSP), neurotoxic (NSP) and amnesic (ASP) shellfish poisoning. Diarrheic shellfish toxicity in the Adriatic Sea has been recorded for the first time in the summer of 1989. The most alarming situation in the Central Adriatic was registered in the eutrophic Kaštela bay. The respective toxins are produced most frequently by dinoflagellates of genus Dinophysis and Prorocentrum. The most important substances of the DSP complex are okadaic acid (OA) and an increasing number of its derivatives named dinophysis toxins (DTX). This class of polyether toxins has received wide interest not only for their unique structure, but also for their pharmacological properties. Threat to public health has been aggravated by a recent discovery that okadaic acid is a highly selective inhibitor of protein phosphatases and a potent tumor promoting agent. The health and economic implications of these events prompted the study of this phenomenon. Control measures to protect humans against DSP contaminated seafood are carried out by application of biological (mouse bioassay), and chemical (HPLC) procedures.

Veliki broj fitoplanktonskih vrsta proizvodi različite toksične metabolite koji se akumuliraju u školjkama što za posljedicu ima intoksikaciju ljudi. Otrovni metaboliti morskih algi mogu se svrstati u 4 skupine: PSP (izazivaju paralizu organa za disanje), DSP (izaziva probavne smetnje), NSP (djeluju na mozak) i ASP (izazivaju amneziju).

The active site accessibility and substrate specificity of the bacterial and mammalian alkaline phosphatases have been investigated using a variety of structurally related alternative substrates, i.e. dialkyl substituted phenyl phosphates. Although large aliphatic side chains do have some influence on substrate binding, the absence of a major steric effect suggests considerable freedom of the leaving group in the ES complex of the human alkaline phosphatases. E.coli alkaline phosphatase exibits restricted binding affinity, indicating speciffic structural differences between the bacterial and the mammalian enzymes. Introduction of an alternative substrate into the reaction system permitted further analysis of the reaction mechanism. The observed deviations from Michaelis-Menten kinetics were investigated in terms of negative cooperative interactions or enzyme asymmetry. Data obtained by non-linear regression curve fitting tend to suggest a mechanism based on independent nonequivalent subunits rather than negative cooperative interactions between subunits of a dimeric enzyme.

Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. Role and mechanism of possible communication between subunits of this dimmeric enzyme are not resolved. Rigid segments in the polypeptide structure supposedly accomplish transfer of conformational information, between the active sites, across the subunit interface. The importance of the subunit interface and the β-pleated sheet, stretching from underneath an active site to the subunit surface, in the catalytic mechanism has been probed by site-directed mutagenesis. The mutation replacing Thr-81 with alanine and Gln-83 with leucine were introduced into the APase gene. Amino acid residues Thr-81 and Gln-83 are located within the β-pleated sheet at the contact surface between the subunits, and form hydrogen bonds with analogous amino acids from the adjacent subunit. Two kinds of mutant proteins were prepared, a single T81A and double T81A/Q83L mutant. Dynamic properties and rigidity of Trp-109 environment of WT APase and mutant enzymes were assessed by acrylamide fluorescence quenching and by measuring phosphorescence lifetime of Trp-109 located in the vicinity of the mutation site and β pleated sheet. Influence of mutations on the kinetic properties determined in 1 M Tris/HCl, pH 8, and in 0.35 M 2A2M1P, pH 10.5 was compared with the effects that mutations had on rigidity of a protein structure.

Non-ribosomal peptide synthetases (NRPS) are molecular machines that synthesize peptide-based natural products, many of which are important pharmaceuticals with antibiotic, immunosuppressor and antitumor activities. A typical NRPS assembly line consists of a number of catalytic modules, each responsible for the incorporation of a single amino acid into the growing peptide product. Modules themselves comprise distinct catalytic domains, such as the adenylation domain (A) that, firstly, selects a specific amino acid monomer to be activated by ATP and, secondly, transfers it to the phosphopantetheinyl arm of the adjacent thiolation domain. Recent biochemical and structural evidence support the idea that the A domain uses a rotational movement of its two subdomains to adapt a single active site for the two partial reactions in the catalytic cycle. It has long been recognized that A domains share a set of 10 conserved motifs (A1-A10), most of which where assigned a particular role in substrate binding and/or catalysis. To our knowledge, this is the first study aimed at elucidating the role of the A9 motif. By analyzing homology models of the A domain from tyrocidine synthetase 1 from Bacillus brevis, it could be observed that during the rotational movement of the subdomains, the A9 motif drastically changes its position relative to the active site. The role of the conserved amino acid residues in this region and its importance for the two partial reactions of the catalytic cycle was examined by site-directed mutagenesis and biochemical characterization of mutant enzymes.

Proantocijanidini iz sjemenki grožđa su polimerni fenolni spojevi koji imaju blagotvoran učinak na čovjekovo zdravlje. Najpoznatiji su po svom antioksidativnom svojstvu, a štite od bolesti tako što smanjuju količine slobodnih radikala u organizmu koji oštećuju stanice. Njihova svojstva su u mnogome određena su stupnjem polimerizacije odnosno brojem monomernih jedinica od kojih su sastavljeni. Ispitana je metoda određivanja stupnja polimerizacije proantocijanidina iz sjemenki grožđa floroglucinolom umjesto benzil merkaptana, koji je izrazito neugodnog mirisa i nezgodan za korištenje. Postupak s floroglucinolom ne zahtjeva rad u digestoru niti posebnu opremu. Uzorci su bile sjemenke autohtonih hrvatskih sorti grožđa Plavca malog, Crljenka i Babića. Uzorci su analizirani korištenjem tekućinske kromatografije visoke djelotvornosti te je iz tih podataka izračunat stupanj polimerizacije proantocijanidina. Uzorci su analizirani korištenjem tekućinske kromatografije visoke djelotvornosti., Proanthocyanidins from grape seeds are polymeric phenolic compounds that have a beneficial effect on human health. They are best known for their antioxidant properties, protecting against disease by reducing the amount of free radicals in the body that damage cells. Their properties are largely determined by the degree of polymerization, that is, by the number of monomer units of which they are composed. The method for determinination of the polymerisation degree of proantocyanidins from grape seeds with phloroglucinol instead of benzyl mercaptan, which has an extremely unpleasant smell and is inconvenient to use, was examined. The procedure with phloroglucinol does not require a digester or special equipment. The samples were seeds of indigenous Croatian grape varieties Plavac mali, Crljenak and Babić. The samples were analyzed using high performance liquid chromatography and the degree of polymerization of proanthocyanidin was calculated from these data.

Jesotoksini su lipofilni, sulfonirani polieteri koji pripadaju skupini morskih fikotoksina, proizvedenih od algi, najčešće dinoflagelata vrste Protoceratium reticulatum, Lingulodinium polyedra i Gonyaulax spinifera. Od prvog otkrića, kada su izolirani iz vrste Patinopecten yessoensis u Japanu, pa sve do danas, njihova prisutnost sve je veća širom svijeta te su s vremenom otkriveni i brojni analozi. Provedena su razna istraživanja i analize na temelju kojih su donekle razjašnjeni mehanizmi djelovanja i toksičnost ovih toksina. Utvrđena je citotoksična aktivnost jesotoksina kroz promjene razina koncentracija kalcija i cAMP-a u stanici, aktivacije kaspaza, otvaranje tranzicijskih pora mitohondrija te modifikacije citoskeleta. Osim toga, djeluju i na imunološki sustav, metabolizam glukoze te imaju zanimljiv utjecaj u liječenju Alzheimerove bolesti. Budući da nisu prijavljeni slučajevi trovanja ljudi, Europska unija povećala je granicu jesotoksina u školjkama s 1 na 3,75 mgYTX/kg školjkaša radi prevencije., Yessotoxins are lipophilic, sulfonated polyethers that belong to the group of marine phycotoxins, produced by algae, most often dinoflagellates of the species Protoceratium reticulatum, Lingulodinium polyedra and Gonyaulax spinifera. From the first discovery, when they were isolated from the species Patinopecten yessoensis in Japan, until today, their presence is increasing all over the world, and over time numerous analogues have been discovered. Various researches and analyzes were carried out, which clarified to some extent the mechanisms of action and toxicity of these toxins. The cytotoxic activity of yesotoxin was determined through changes in the cellular level of calcium and cAMP, caspase activation, opening of mitochondrial transition pores and modifications of the cytoskeleton. In addition, it also affects the immune system, glucose metabolism and has an interesting effect in the treatment of Alzheimer's disease. Since no cases of human poisoning have been reported, the European Union increased the limit of yesotoxin in shellfish from 1 to 3.75 mgYTX/kg of shellfish for prevention.

Morski plodovi čine važnu sastavnicu uravnotežene ljudske prehrane, ali njihovom konzumacijom može doći do intoksikacije ukoliko su kontaminirani fitoplanktonskim toksinima. Proizvode ih fitoplanktonske alge, a nakupljaju se u morskim organizmima koji se hrane fitoplanktonom. Fitoplanktonski toksini su skupina kompleksnih organskih spojeva, a podijeljeni su u šest skupina s obzirom na simptome trovanja koje izazivaju. Pored negativnih utjecaja na ljudsko zdravlje kao bioaktivne molekule imaju i potencijala u razvoju novih lijekova. Zabrinjavajuća činjenica je što s porastom temperatura mora i oceana dolazi do povećane produkcije ovih toksina koji se pojavljuju i na mjestima gdje ih do sad nije bilo. Analiza uzorka školjkaša provedena je s ciljem detekcije i identifikacije poznatih i novih vrsta toksina na području Jadranskog mora. Kao metoda se koristila tekućinska kromatografija uparena s masenom spektrometrijom visoke rezolucije., Seafood is an important component of a balanced diet, but their consumption can lead to intoxication if they are contaminated with phytoplankton toxins. They are produced by phytoplankton algae and accumulate in marine organisms that feed on phytoplankton. Phytoplankton toxins are a group of complex organic compounds divided into six groups according to the symptoms they cause. In addition to negative effects on human health, as bioactive molecules they also have potential in the development of new drugs. The worrying fact is that the rise in sea and ocean temperatures leads to an increased production of these toxins, which appear in places where they were not detected before. The analysis of the shellfish sample was carried out with the aim of detecting and identifying known and new types of toxins in the Adriatic Sea. The method used was liquid chromatography coupled with high-resolution mass spectrometry.

Proantocijanidini su kondenzirani tanini koji se nalaze u sjemenkama i kožici grožđa, a u zadnje vrijeme česti su predmet istraživanja zbog pozitivnog djelovanja na ljudski organizam. Imaju ulogu u prevenciji raka i kardiovaskularnih bolesti, a upravo od te dvije bolesti umire najviše ljudi. Cilj ovog rada bio je razdvojiti proantocijanidine sjemenki grožđa s obzirom na broj jedinica od kojih su građeni na HPLC uređaju s kolonom normalne faze te odrediti stupanj polimerizacije proantocijanidina. Uspješno smo razdvojili proantocijanidine do stupnja polimerizacije n=7 te iz dobivenih rezultata smo izračunali stupanj polimerizacije. Koristeći kiselo kataliziranu reakciju tiolize s benzil merkaptanom na HPLC uređaju s kolonom reverzne faze razdvojili smo benzil tioetere katehina, epikatehina i epikatehin galata. Iz tih podataka smo također izračunali stupanj polimerizacije proantocijanidina. Stupnjevi polimerizacije dobiveni na dva načina pokazuju dobro slaganje., Proanthocyanidins are condensed tannins which are mainly found in grape seeds and skins. In the last few years proanthocyanidins have been extensively researched because of their beneficial effects on human health, they show great results in preventing cancer and cardiovascular diseases. The goal of this research is to separate grape seeds proanthocyanidins on normal phase HPLC and to determine the degree of polymerization of proanthocyanidins using two methods. We have successfully separated polymers up to n=7 and calculated the degree of polymerization using information from chromatograms. Analyzing products of acid-catalysed thiolysis reaction with benzyl mercaptan on reverse phase HPLC we have successfully separated benzyl thioethers of catechin, epicatechin and epicatechin gallate. Information from chromatograms were used to determine the degree of polymerization. The degrees of polymerization calculated in both ways coincide.

Zmijski otrov je izlučevina preoblikovanih žlijezda slinovnica. Sadrži dvadesetak tvari. Više od 90% suhog zmijskog otrova čine proteini. Neki od njih su toksični, a neki ne pokazuju toksična svojstva. Jedan od toksina je α-kobra toksin. α-Kobra toksin spada u α-neurotoksine s dugim lancem, a pronalazimo ga kod vrsta roda Naja. α-Kobra toksin antagonist je nikotinskog acetilkolinskog receptora (nAChR). Vezanjem na nAChR mišićnog i neuronskog tipa, blokira sposobnost receptora da veže acetilkolin. Na taj način inhibira protok iona kroz postsinaptičku membranu što dovodi do paralize i prestanka disanja. Kod ugriza otrovnica roda Naja najčešće se primjenjuju polivalentni protuotrovi., Snake venom is an excretion of transformed salivary glands and contains about twenty substances. More than 90% of dry snake venom consist of proteins. Some of them are toxic and some do not show toxic properties. One of the toxins is α-cobra toxin. α-Cobra toxin belongs to α-neurotoxins with a long chain, and it is found in a species of the genus Naja. α-Cobra toxin is an antagonist of the nicotinic acetylcholine receptor (nAChR). By binding to muscle and neuronal type nAChR, it blocks the receptor's ability to bind acetylcholine. In this way it inhibits the flow of ions through the postsynaptic membrane, leading to paralysis and cessation of breathing. Polyvalent antivenoms are most often used in the case of bites by venomous snakes of the genus Naja.