A collagenolytic enzyme fraction was isolated from the hepatopancreas of the lobster (Homarus americanus) and semi-purified by the successive steps of acetone precipitation, ammonium sulfate fractionation, ion exchange chromatography on Mono Q column, followed by gel filtration on a Superdex 75 column or by preparative isoelectric focusing using a Rotofor cell. / Semi-purified collagenase fractions from the lobster hepatopancreas was electrophoresed in polyacrylamide gels both in the presence or absence of SDS, and shown to have molecular weights ranging from 15,000-66,000. The enzymatically active peak 1 fraction from the isoelectric focusing step in the Rotofor cell migrated as a single band in 12% polyacrylamide gel with few light protein bands. / The pH-activity data indicated that the collagenase fraction had two pH optima for the hydrolysis of native collagen, one at pH 4 and the other between pH 7-8. / The temperature-activity data for the hydrolysis of native collagen indicated the lobster enzyme exhibited two temperature optima--a minor one at 25$ sp circ$C and a more pronounced one between 40$ sp circ$C and 50$ sp circ$C.

Collagenases were extracted from the skeletal muscle of winter flounder (Pseudopleuronectes americanus) with Tris-HCl buffer, pH 7.4, containing 5 mM CaCl$ sb2.$ The crude extract in the active form was purified by ammonium sulfate fractionation, followed by a succession of column chromatographic steps which included ion-exchange, immobilized metal affinity and size-exclusion in the Fast Protein Liquid Chromatography (FPLC) system. The trypsin-like and chymotrypsin-like activities of the crude extract diminished with purification. A comparative study of the collagenase fraction from ion-exchange chromatography (IEX-1) and the commercial collagenase fraction from Clostridium histolyticum indicated that the two enzymes were similar with respect to their response to temperature but differed with respect to their response to pH. The enzymes differed slightly in terms of their thermal and pH stabilities. The winter flounder collagenase fraction from size-exclusion chromatography (SEC) had a higher optimum pH temperature than the IEX-1 fraction as well as the commercial collagenase. However, both SEC and the IEX-1 extracts had the same optimum pH. The collagenase fraction from SEC had a slightly lower thermal stability than the IEX-1 fraction and the commercial collagenase. / The higher catalytic efficiency (V$ sb{ rm max}$/K$ sb{ rm m}$') and the lower $ Delta$G values for C. histolyticum collagenases showed that bacterial collagenases are better catalysts than winter flounder skeletal muscle collagenases for the PZ-peptide hydrolase reaction at 37$ sp circ$C and pH 7.1. / Zymography revealed the presence of two collagenase isoenzymes from the fish muscle, designated as WFC-1 and WFC-2 with molecular weights of 79,600 and 75,500, respectively. WFC-1 was separated from WFC-2 by electrophoretic blotting onto the PVDF membrane. The amino acid composition of WFC-1 and WFC-2 were closely related. / The fish collagenases were inhibited by metal chelators, EDTA and 1,10-phenanthroline suggesting that these enzymes are metalloproteases. The enzyme activity in the presence of EDTA and 1,10-phenanthroline were recovered upon addition of low levels of calcium and zinc ions, respectively. Higher levels of these metal ions inhibited the isoenzymes. 2-Mercaptoethanol and dithiothreitol were also effective inhibitors.

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Bibliography: leaves 205-233 / xix, 234 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.S.)--University of Adelaide, 1985

A polarimetric method for monitoring the rate of soluble collagen breakdown by collagenase enzyme action has been developed. The method represents an extension of previous physicochemical techniques based on viscometry, but is simpler and easier to carry out, particularly in the case of reaction rate studies. The method was developed arising from reports of collagenase activity measurement on inappropriate substrates such as gelatin, modified collagens and synthetic polypeptides. The optical method depends on measurement of the loss in optical rotation in solutions of soluble calfskin collagen resulting from initial enzymic cleavage of the collagen trip1e-helix, followed by spontaneous unwinding of the resultant unstable helical fragments. Specific assay conditions were chosen to ensure that the loss in optical rotation following enzymic cleavage was rapid and complete. The method is specific since in the absence of collagenase, non-specific proteinases produce only a limited decrease in solution optical activity. The method has also been compared with established physicochemical assay techniques and compares favourably with both viscometric and titrimetric collagenase assays. The availability of a rapid, sensitive and quantitative procedure for measurement of collagenase activity provides a convenient means for detecting the presence of collagenase in solution and examination of hide bacterial cultures for collagenase production. In addition, a study of biocidal compounds of potential interest in hide preservation for possible inhibitory effects on collagenase is conveniently carried out with the method. Fundamental research into synergistic action in enzymic hydrolysis of collagen is now possible, providing valuable insight into the mechanism of raw hide biodeterioration.

In the leather industry fungicides are often used for the protection of wet-blue leather. These fungicides are usually only sparingly soluble and are therefore formulated together with surfactants in order to increase their solubility and to ensure an even distribution over the surface of the hide after treatment. Solutions containing both fungicides and surfactant are complex. The nature of these solutions was investigated. By means of UV/Vis spectroscopy and viscometry it was shown that the surfactant and fungicides form micelles and mixed micelles in solution. The nature of these micelles and mixed micelles was dependent on the solution temperature as well as on the concentrations of the surfactant and fungicides. At the higher temperatures and concentrations transition to large, possibly rod-shaped, mixed micelles occurred. The interaction between the selected fungicides 2-(thiocyanomethylthio)benzothiazole and n-octyl-4-isothiazol-3-one with bovine skin collagen in the form of both limed and lightly chromed hide powder in the presence of the non ionic surfactant Triton X -100 was investigated. Fungicide uptake was determined by difference measurements on the float solutions at regular intervals during treatment. Binding was rapid with equilibrium being established within the first six hours even for the solutions with the highest surfactant concentration. Binding failed to follow a normal mass-action binding-type isotherm approaching a saturation limit, but increased continuously indicating a co-operative effect whereby binding site affinity actually increased with the amount of ligand bound. Binding was accompanied by a drop in the free surfactant in the solution at the higher biocide levels indicating the formation of complex mixed micelles which bind to the collagen fibres. The uptake and antifungal activity of commercial fomulations of the fungicides on chrome-tanned wet-blue leather was investigated at various treatment temperatures. At lower fungicide treatment concentrations, binding tended to follow a typical mass-action type binding isotherm, increasing slightly with temperature. At higher float concentrations, an inflexion point was apparent beyond which uptake showed a marked increase with concentration. This inflexion point, signifying a change in binding characteristics, occurred at progressively lower concentrations with increasing temperature. Antifungal activity in terms of storage periods to onset of fungal growth was determined on the wet-blue leather cuttings immediately after treatment and drainage and also on sample discs after exhaustive extraction of free fungicide using dichloromethane. Storage performance testing of the various treated wet-blue leathers was carried out by different methods. Residual protective periods showed a curvilinear increase with dosage offer and surface uptake. In the low dosage range treatment temperature had only a relatively slight effect in promoting uptake and improving storage protection. At higher dosages, the influence of temperature on uptake and storage protection was greater due to the increase in surface binding of the fungicides at the elevated temperatures. Only a portion of the fungicide uptake was recovered by direct solvent extraction of the treated wet-blue leather. Solvent extraction reduced storage margins. The storage response in relation to fungicide content was, however comparable after extraction, indicating that both irreversibly bound and physically associated fungicide offered effective protection. Results of the study provide further insight into the mode of interaction of fungicide emulsion dispersion with bovine skin collagen, and the importance of the emulsion dispersions and its stability in determining the uptake of fungicide.

A leptospirose é uma zoonose, amplamente difundida pelo mundo, causada por espiroquetas patogênicas do gênero Leptospira, que colonizam os túbulos renais de animais silvestres e domésticos. A transmissão ocorre, principalmente, pelo contato direto com água e solo contaminados com a urina de animais infectados que podem ser clinicamente assintomáticos. As leptospiras patogênicas invadem os tecidos do hospedeiro através da penetração da pele lesada ou mucosas da boca, narina e olhos. Logo após ultrapassar as superfícies de contato, as bactérias chegam rapidamente à corrente sanguínea e espalham-se para todos os órgãos causando lesões, principalmente, no fígado e rins onde produzem hemorragia e necrose tecidual. Após a entrada no hospedeiro, a progressão da infecção envolve a adesão das bactérias às células eucarióticas e às proteínas de matriz extracelular seguida pela invasão aos tecidos. Estudos recentes demonstraram que as leptospiras são capazes de se translocarem através das monocamadas celulares, o que poderia ser um mecanismo de evasão do sistema imune e também facilitaria a entrada e saída da corrente sanguínea para infectar órgãos-alvo. O mecanismo envolvido na invasão do patógeno através das barreiras extracelulares não está bem elucidado. Enzimas capazes de degradar proteínas da matriz extracelular poderiam contribuir com a motilidade e quimiotaxia das bactérias durante a invasão. Bactérias patogênicas sintetizam e secretam diferentes tipos de proteases, que atuam degradando colágeno e glicoproteínas entre outras proteínas do hospedeiro. Recentemente, um estudo, utilizando gelatina e caseína como substratos e lisado bacteriano, mostrou haver uma variedade de proteases em Leptospira spp. Análises do genoma indicam a presença de vários genes que codificam prováveis proteases. A comprovação experimental da existência e a caracterização funcional destas proteínas poderão contribuir no entendimento da patogenia da leptospirose. Neste sentido, este trabalho teve como objetivos a clonagem, expressão e caracterização funcional de uma provável colagenase (ColA) de L.interrogans sorovar Copenhageni. As sequências codificantes do domínio de colagenase 1 (D1), do domínio de colagenase 2 (D2) e de ambos os domínios (Full) da ColA foram amplificadas por PCR a partir de DNA genômico de Leptospira e clonadas no vetor de expressão pAE. Os fragmentos D1, D2 e Full da ColA foram expressos em E. coli BL21 - SI e purificados a partir das frações insolúveis por cromatografia de afinidade a níquel. Os fragmentos recombinantes purificados foram utilizados na obtenção dos antissoros policlonais, e as atividades enzimáticas de cada um foram avaliadas. Os antissoros policlonais produzidos em coelho apresentaram elevados níveis de anticorpos detectados por ELISA. Experimentos de Western - blotting demonstraram a presença de proteína ColA em diferentes sorovares patogênicos de Leptospira spp. As proteínas Full e D2 apresentaram atividade catalítica sobre o colágeno desnaturado e sobre peptídeo sintético e atividade hemorrágica em camundongos. Estes resultados indicam que ColA é provavelmente uma proteína de leptospira envolvidas na invasão de tecidos do hospedeiro. / Leptospirosis is a zoonosis widespread throughout the world, caused by pathogenic spirochetes of the genus Leptospira, which colonize the renal tubules of wild and domestic animals. Transmission occurs mainly through direct contact with water and soil contaminated with urine of infected animals that may be clinically asymptomatic. Pathogenic leptospires invade host tissues by penetrating damaged skin or the mucous membranes of the mouth, nostrils and eyes. Soon after passing the contact surfaces, leptospires come quickly into the bloodstream and spread to all organs causing damage mainly in the liver and kidneys where they produce hemorrhage and tissue necrosis after entering the host, the progression of the infection involves the adhesion of bacteria to eukaryotic cells and extracellular matrix proteins followed by invasion of tissues. Recent studies have shown that leptospires are able to translocate across cell monolayers, which could be a mechanism for evasion of the immune system and also facilitate the entry and exit from the bloodstream to infect target organs. The mechanism involved in pathogen invasion through extracellular barriers is not well elucidated. Enzymes capable of degrading extracellular matrix proteins could contribute to motility and chemotaxis of bacteria during the invasion. Pathogenic bacteria synthesize and secrete different types of proteases that degrade collagen and glycoproteins among other host proteins. Recently, a study using gelatin and casein as substrates and bacterial lysate showed a variety of proteases in Leptospira spp. Analysis of the genome indicate the presence of several genes encoding probable protease. The experimental proof of the existence and functional characterization of these proteins may contribute to the understanding of the pathogenesis of leptospirosis. In this sense, this work aimed the cloning, expression and functional characterization of a probable collagenase (ColA) from L.interrogans serovar Copenhageni. Coding sequences of the collagenase domain 1 (D1), collagenase domain 2 (D2), and both domains (Full) of the ColA gene were amplified by PCR from genomic Leptospira DNA and cloned into the pAE expression vector. The D1, D2 and Full fragments of ColA protein were expressed in E. coli BL21-SI and purified from the insoluble fractions by nickel affinity chromatography. The purified fragments were used to obtain the polyclonal antiserum, and their enzymatic activities were evaluated by zymography. Rabbit polyclonal antiserum against the recombinant protein fragments were produced with a high antibody level detected by ELISA. Western-blotting experiments demonstrated the presence of ColA protein in different pathogenic serovars of Leptospira. The Full and D2 proteins showed catalytic activity on denatured collagen and synthetic peptide and hemorrhagic activity in mice. These results indicated that ColA is probably a leptospiral protein involved in invasion of host tissues.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2001. / Bibliography: leaves 184-201.