Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

The proteolytic system of Lactococcus lactis starter cultures influences both flavor and the characteristic body and texture of cheese. The ability to further understand and control how different components of this proteolytic system work together to hydrolyze milk proteins would be of immense importance to the dairy industry. The goal of this research was to characterize Lactococcus lactis subsp. lactis starter bacteria with varying prt operon compositions by proteinase specificity, aminopeptidase and lipase activities, growth, and influence on cheese flavor. By using a cheese slurry system, a statistical model to predict milk protein hydrolysis patterns was developed. Lactococcus lactis subsp. lactis C20 has five plasmids of 55 (pJK550), 48 (pJK480), 43 (pJK430), 3.7 (pJK037), and 2.1 (pJK021) kilo bases. Two of these plasmids (pJK550 and pJK430) are necessary for full proteolytic capability, i.e., clotting milk in 16 h at 20°C. Plasmid pJK550 codes for a proteinase that catalyses the first step in casein degradation. Plasmid pJK430 codes for an oligopeptide transport system, which further transports peptides across the membrane for bacterial metabolism. Strains were constructed containing twelve different combinations of proteolytic phenotypes, such as Lac+PrtP+Opp+, Lac+PrtP+Opp-, Lac+PrtP-Opp+, Lac+Prt-Opp-, Lac-PrtP+Opp+, Lac-PrtP+Opp-, Lac-Prt-Opp+, and Lac-Prt-Opp-. The proteinase specificities of these strains toward milk proteins were dependent on the genotypes present. Genetically all strains showed a P1-type proteinase. Enzymatically C20 had group g proteinase specificity, whereas the rest of the strains containing the proteinase gene showed mixed group specificity. a a-Casein was only slightly hydrolyzed by all strains. B-Casein had a variable pattern, as did mixed casein and milk. K-Casein hydrolysis showed similar degradation patterns in all strains except CB06, which varied in its profile from the other strains. Sensory evaluation showed that culture had a significant effect on rancidity but not on acidity or bitterness. It also showed that the proteolytic system was associated with lipase activity in these strains. A statistical prediction model was developed that allowed strains to be classified according to their amino acid hydrolysis patterns. Mixed casein solution proved to be the best substrate for this analysis. Relationships among strains were seen more easily with canonical analysis and distance tables than by looking only at amino acid hydrolysis patterns.

A series of experiments were conducted to investigate the effects of adding an exogenous proteolytic enzyme (EPE) on the growth performance of beef steers fed growing and finishing diets containing 30% dried distillers grains with solubles (DDGS; Exp. 1), and results corroborated by in vitro ruminal fermentation in continuous cultures (Exp. 2). In Exp. 1, 48 group-penned Angus crossbred steers were randomly assigned to 2 treatments (n = 6) in a completely randomized design: DDGS TMR (DT) without and with EPE (27 mg of azocasein hydrolyzed/min/kg DM TMR). The addition of EPE during the growing phase increased DMI (P = 0.02), but had no effects on final BW, BW change, ADG, and G:F. Adding EPE during the growing phase decreased NDF digestibility, whereas the digestibility of DM, CP, and ADF were not affected. There was a tendency for both ADG (P = 0.09) and final BW (P = 0.11) to increase during the finishing phase without affecting BW change and G:F. As opposed to the growing phase, EPE increased digestibility (P < 0.04) of DM, CP, NDF, and ADF. In Exp. 2, 4 dietary treatments were assessed in continuous cultures; non-DDGS TMR (NDT) or DT finishing beef steer diet was combined without or with EPE in a 2 × 2 factorial design. The DT was the same diet used as the finishing diet in Exp. 1, and dose rate of EPE was the same as Exp. 1. Feeding the DT increased total VFA concentration (P = 0.01) which corresponded with a decreased (P < 0.01) pH compared with the NDT diet (5.8 vs. 6.0) regardless of EPE supplementation. Supplementing EPE tended to increase (P = 0.07) the total VFA concentration in both diets, but only increased digestibility of DM, OM, and NDF when added to the DT diet (P < 0.05), leading to tendencies on TMR × enzyme interaction (P < 0.10). Addition of the EPE product assessed in this study resulted in positive responses in Exp. 1 and 2 when added to finishing beef steer diets, and thus it is clear that use of protease enzyme products may be more effective in high concentrate diets such as finishing beef steer diets containing DDGS.

This work reviews studies of bovine K-casein and specifically K-casein macropeptide. Properties of K-casein, its structure and heterogeneity, proteolytic activity of some milk clotting enzymes on K-casein, and K-casein sensitive bonds are discussed. Macropeptides of other species are also presented. The carbohydrate moieties of bovine macropeptide together with their biological and physiological functions are reviewed. Macropeptides were produced by enzymic hydrolysis from whole casein solution using crystalline chymosin (EC 3.4.23.4). Trichloroacetic acid (final concentrations 2, 8 and 12%) was added after 5, 30 and 60 min of incubation to precipitate protein and inactivate the enzyme. The filtrate was then exhaustively dialyzed against distilled water to remove trichloroacetic acid and small molecules. The dialyzate was lyophilized and stored at -20deg;c until required for analysis. These macropeptides were then compared using RP-HPLC with macropeptides obtained from purified K-casein isolates by the same method (15 min incubation). Proteolytic activity of some milk-clotting enzymes (chymosin, Mucor miehei rennet and Endothia parasitica rennet) and some proteinases (trypsin and chymotrypsin) on K-casein and macropeptide isolated from K-casein was followed by RP-HPLC. The milk-clotting enzymes were standardized to the same clotting activity using a Formagraph. Each enzyme was incubated with .5 mix-casein and macropeptide solutions (10 mg in 1 ml .05 MpH 6.6 phosphate buffer) at 37°C for various incubation times. Reactions were stopped by addition .5 ml of 8 Murea containing 10-5 Mpepstatin or .025 ml pepstatin (1 mg pepstatin in 1 ml methanol). These reaction mixtures were separated into fractions using RP-HPLC and chromatograms of the different enzymes compared.

No description available.

Orala bakterier, såsom de tidiga kolonisatörerna; Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis och Actinomyces naeslundii uttrycker ett brett spektrum av ytadhesiner som möjliggör inbindning till receptorer i tandpellikeln. Saliv, gingivalt exudat (GCF) och kollagen I i cement på blottade rotytor erbjuder möjliga ytor i munhålan där bakterier kan adherera och bilda biofilm. Syftet med denna studie var att undersöka huruvida utvalda bakterier kan förändra genuttryck beroende på innehållet i olika ytor. Laborativa in vitro-försök genomfördes där de fyra bakteriearterna tilläts växa på ytor täckta med de tre olika substrat; saliv, serum och kollagen I. Graden av inducerad proteolytisk aktivitet, yt-associerad såväl som utsöndrad, uppskattades därefter med hjälp av ett FITC-konjugerat substrat, radiell diffusionsteknik samt spektrofotometri. Enligt studiens hypotes skulle bakteriearterna anpassa sig beroende på ytan de fäste till, och därigenom ändra metabol aktivitet såsom proteasuttryck. Baserat på resultaten kunde små förändringar noteras. Dock kunde inga bestämda slutsatser dras vad gäller förändrad proteolytisk förmåga hos de utvalda bakterierna exponerade för de olika orala ytorna i studien. / Oral bacteria, such as the early colonizers; Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Actinomyces naeslundii display a wide range of surface adhesins which enable them to bind to receptors in the tooth pellicle. Saliva, gingival crevicular fluid (GCF) and collagen I in cementum on uncovered root surfaces present possible binding sites in the oral cavity onto which microorganisms can adhere and form a biofilm. The aim of this study was to assess whether these selected bacteria can alter their gene expression in response to protein components found on the various surfaces. In vitro laboratory assays were conducted, where the four oral species were added to surfaces coated with three substrates; human saliva, human serum and collagen I. The degree of induced proteolytic activity, surface-associated as well as secreted, was subsequently assessed using a FITC-labelled protease substrate, radial diffusion assays on skim milk agar and spectrophotometry. The hypothesis underlying the study was that bacterial species adapt depending on the surfaces they adhere to, thus altering protease expression. Based on the results, small variations could be detected, although no firm conclusion can be drawn regarding proteolytic abilities of the selected bacteria when exposed to the surfaces tested here.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / ADAMs (a disintegrin and metalloprotease) can influence multiple cellular processes involved in normal development and pathogenesis. ADAM12 expression levels are elevated in many pathological conditions including cancer, cardiovascular disease, and muscle regeneration. Recently, ADAM12 has emerged as a candidate cancer gene in a comprehensive genetic analysis of human breast cancers. The regulation of ADAM12 expression is poorly understood. Identification of new substrates for ADAM12 metalloprotease can expand our knowledge of processes in which ADAM12 is involved. Here, we show that ADAM12 expression is upregulated by transforming growth factor beta (TGF-beta), an essential signaling pathway for many cellular processes. This upregulation requires proteosomal degradation of a transcriptional repressor SnoN. Furthermore, breast cancer cell lines expressing high levels of SnoN have significantly impaired induction of ADAM12 by TGF-beta, suggesting an inverse correlation between SnoN and the extent of regulation of ADAM12 by TGF-beta. Additionally, we demonstrate that ADAM12 is one of the metalloproteases involved in shedding a Notch ligand, Delta like 1 (Dll1). The Notch signaling pathway plays a crucial role in cell fate decision during development and in adults. Cleavage of Dll1 by ADAMs occurs in cis and results in activation of Notch signaling in a cell-autonomous manner. Furthermore, the intracellular domain of Dll1 created after cleavage further enhances TGF-beta signaling in response to TGF-beta. Our analysis of breast cancer-associated mutations in the ADAM12 gene showed a lack of proper proteolytic processing of the ADAM12 protein and its mislocalization to the endoplasmic reticulum. Additionally, ADAM12 mutants show a dominant-negative effect on the processing of the wild-type ADAM12 and result in loss of the functional ADAM12 at the cell surface. Collectively, our results indicate a new mechanism of regulation of ADAM12 expression, expand the role of ADAM12 in the regulation of Notch signaling, and characterize cancer-associated mutations in the ADAM12 gene.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

A Bothrops atrox é uma serpente de amplia distribuição na Sul América e é responsável por um número importante de mortes de pessoas, principalmente na Amazônia. As alterações na composição do veneno desta espécie têm sido associados a fatores como a ontogenia, distribuição geográfica e alimentação. Assim, este projeto visa comparar e identificar a partir da diferença entre os sexos, as características bioquímicas e biológicas do veneno de irmãos de B. atrox, sob condições ambientais controladas, contribuindo no conhecimento das mudanças nas características do veneno da espécie e pudendo auxiliar no aprimoramento da produção de antissoros mais efetivos. Os venenos foram coletados de 5 fêmeas e 4 machos irmãos de B. atrox, nascidas em cativeiro. Os venenos foram analisados quanto individualmente como o pool de cada grupo. As análises consistiram em dosagem de proteína através de BCA, eletroforese mono e bidimensional, cromatografia liquida, espectrometria de massas, atividades caseinolítica, fosfolipásica A2, L-aminoácido oxidase, zimografias contendo gelatina e caseína como substrato, dose mínima coagulante sobre o plasma e fibrinogênio, dose leta 50% e dose mínima hemorrágica. A análise individual dos venenos mostrou que os machos apresentaram maior concentração de proteínas e atividade fosfolipásica A2. No quanto aos pools de veneno, o das fêmeas apresentou maior letalidade e capacidade coagulante sobre plasma e fibrinogênio e o dos machos apresentaram maior capacidade hemorrágica e atividade L-aminoácido oxidase. O perfil espectrométrico mostrou que o pool de veneno das fêmeas, teve um 29% a mais na quantidade de proteínas identificadas em relação aos machos. Em conclusão, a ação do veneno das fêmeas estaria relacionado a uma maior capacidade para gerar dano sistêmico na presa, entanto que os venenos dos machos poderiam ocasionar um maior dano local. Além, a variabilidade nas atividades biológicas dos venenos confirma que além dos fatores ambientais existem outros que poderiam influir na plasticidade da composição dos venenos. / Bothrops atrox snake is widespread in South America and causing a large number of human deaths, mainly in the Amazon. Changes in the composition of the venom of this species have been linked to factors such as ontogeny, geographical distribution and feeding. Thus, this study aims to compare and identify from the sex difference, the biochemical and biological characteristics of venom of B. atrox siblings, under controlled environmental conditions, contributing to the knowledge of changes in the characteristics of the venom of the species and can assist in improving the production of more effective antisera. Venoms were collected from 5 females and 4 males of B. atrox siblings, born in captivity. The venoms were analyzed both, individually and as a pool of each group. The assays consisted in protein quantification using BCA, one and two-dimensional electrophorese, liquid chromatography, mass spectrometry, caseinolytic, phospholipase A2, and L-amino acid oxidase activities, zimography containing gelatin and casein as substrate, minimum coagulant dose upon plasma and fibrinogen, lethal dose 50 % and minimum hemorrhagic dose. Individual analysis of venoms showed that males had higher proteins concentration and phospholipase A2 activity. Concerning the venoms pool, the female showed higher lethality and coagulant capacity upon plasma and fibrinogen and the male had higher L-amino acid oxidase activity and hemorrhagic capacity. Spectrometric profile showed that the venom pool of female snakes had a 29 % increase in the number of proteins identified in comparison to males. In conclusion, the action of the female venom would be related to a higher capacity to generate systemic damage in the prey and male venoms could lead to higher local damage. In addition, variability in the biological activities of venoms confirms that there are other factors that could would be influencing the plasticity of the composition of venoms, in addition to environmental.