No description available.

Peroxidase enzymes are of critical importance within the scientific community for their applications in biosensing assays. In a living system, natural peroxidases function as catalysts in the oxidation of peroxide (e.g., H2O2) - a harmful byproduct of aerobic processes and convert them into harmless compounds. Such an ability allows peroxidases to serve as labels in biosensing assays, where they are conjugated to antibodies and accurately produce a detection signal by catalyzing substrates. However, due to intrinsic limitations, namely instability, Peroxidase made of proteins substantially inhibit broader applications. Alternatively, nanoparticles produced from noble metals have been found to exhibit peroxidase-like abilities and, therefore, can be used as synthetic enzymes with the potential to replace their natural counterparts. Given that the stability of most peroxidase mimics is already much better than their natural counterparts, in this field, the principal challenge has been creating substantial improvements to the catalytic efficiency of the mimics. This study sought to create a cage-like nanostructure ( denoted as nanocages) consisting of two platinum group metals. This experiment uses Galvanic replacement as a mechanism to hollow all Nanocages formed. Galvanic replacement has been primarily demonstrated using coinage metals ( Ex. Ag and Au). This experiment seeks to show that this process is viable for other Nobel metals, as well. In particular, palladium cubes were used as scaffolds or sacrificed templates to induce the reaction with a precursor containing a secondary Nobel metal (Platinum, Rhodium, or Ruthenium). Once viable samples where produced and checked via TEM ( Transmission Electron microscope), the peroxidase-like activity was compared to the activity of a non-hollowed nanostructure of the same material composition using TMB colorimetric assay.

No description available.

No description available.

No description available.

The purpose of this research was to characterize the antibody-mediated inhibition of horseradish peroxidase activity. Inhibition of enzyme activity was determined when alternate substrates were used. Typical biphasic patterns of inhibition were observed with each of five electron donors and three hydroperoxides. In no case was inhibition complete (100%). Inhibition assays with varied substrate concentrations revealed a dependence of anticatalytic activity upon the hydroperoxide concentration. Increased hydroperoxide concentration elevated inhibition levels. Inhibition is not dependent upon the electron donor concentration. The dependence of inhibition levels on hydroperoxide concentration suggests that antibody may make the horseradish peroxidase basic isoenzyme more susceptible to peroxide inactivation. Kinetic data show increased enzyme affinity for hydrogen peroxide in the presence of antibody. In addition, acidic peroxidases that are less susceptible to peroxide are poorly inhibited by antibasic horseradish peroxidase. Spectrophotometric experiments with an electron donor analogue indicated that antibody does not inhibit horseradish peroxidase activity by preventing electron donor binding. Inhibitory antibody binding sites could not be determined because fragments of the enzyme capable of binding inhibitory antibody could not be produced. Antisera prepared against acidic horseradish peroxidase isoenzymes were used in immunological procedures (precipitation, anticatalytic assays) to assess the homology of horseradish, radish and turnip peroxidases. Acidic horseradish peroxidases with similar physicochemical properties are distinguishable by immunological methods. Interspecies relationships are stronger among acidic peroxidases than relationships between acidic and basic horseradish isoenzymes. These results support an evolutionary conservation of acidic isoenzymes and suggest that each isoenzyme group catalyzes specialized plant functions. / M.S.

The effects of weaning stressors on several immune and health responses were measured in three experiments. Sixty-four beef calves from 2 sources were bled on d - 7 (pre-weaning), 0 (weaning), 7, 14, and 21 (post-weaning). Serum selenium (Se), neutrophil and lymphocyte counts, and glutathione peroxidase (GSH-Px) activity for erythrocytes and leukocytes were measured for each calf at each date. Erythrocyte GSH-Px activity remained constant over time, while leukocyte GSH-Px and serum Se increased on d 7 (P<0.0001). Effects of single mineral vs. multi-mineral supplementation were measured for several immune and health responses in 2 trials. Trial 1: 36 heifers weaned on-site at SVAREC were randomly assigned 1 of 2 dietary supplements: 1) no supplement; 2) 15% CP at 0.5% BW; and 1 of 3 injection treatments: 1) no treatment (control); 2) Mu-Se injection; 3) Multi-Min injection. Whole blood Se and serum Cu increased post-weaning and serum Zn decreased post-weaning. Mu-Se-supplemented heifers gained weight faster between d 14-28 vs. Multi-Min-supplemented (P=0.01) or control heifers (P=0.02). Trial 2: 48 steers purchased at auction and transported to SVAREC were randomly assigned to 1 of 4 pasture management systems: 1) control (no treatment); 2) litter fed; 3) litter applied; 4) inorganic fertilizer; and 1 of 3 injection treatments (same as Trial 1). Whole blood Se and serum Cu increased post-stress and serum Zn decreased post-stress. Oxidative burst activity decreased in Mu-Se and Multi-min supplemented steers between d 0-4 vs. control steers (P<0.01). Multi-min-supplemented steers had higher phagocytic activity vs. steers in either Mu-Se or control groups (P=0.04). / Master of Science

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study describes: a) the isolation and purification of horseradish peroxidase isoenzymes from horseradish roots, b) the characterization of various forms and components of the enzyme by cation-exchange and reversed-phase high performance liquid chromatography, c) the preparation of antibodies against horseradish peroxidase isoenzymes, d) immunological studies for the development of an isoenzyme quantification method and e) the formation of an enzyme-melamine conjugate for use in a melamine quantification immunoassay. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die isolering en suiwering van peperwortel-peroksidase-isoënsieme vanuit die peperwortel, b) die karakterisering van verskillende vorme en komponente van dié ensiem deur katioonuitruilings en omgekeerde-fase HPLC c) die voorbereiding van teenliggaampies vir peperwortel-peroksidase-isoënsieme, d) immunologiese studies vir die ontwikkeling van 'n isoënsiem-kwantifiseringsmetode; en e) die vorming van 'n ensiem-melamien-konjugaat vir gebruik in 'n melamienkwantifiseringsmetode.

Xylella fastidiosa é uma bactéria gram-negativa, colonizadora do xilema de plantas economicamente importantes, sendo responsável por diversas patogenias como a doença de Pierce em videiras e a clorose variegada dos citros (CVC). Plantas, ao serem infectadas por patógenos, dispõem de um maquinário de defesa que inclui a geração de espécies reativas de oxigênio (ROS). Peróxidos de lipídios podem ser formados pelo ataque de ROS à membrana bacteriana ou pela ação de lipoxigenases. O sistema da AhpR (alquil hidroperóxido redutase) foi inicialmente caracterizado como o principal responsável pela defesa contra hidroperóxidos orgânicos em bactéria. Recentemente, foi descrito um gene em muitas bactérias patógenas no qual a sua deleção conferia a célula uma maior susceptibilidade a hidroperóxidos orgânicos, mas não a H2O2 ou a geradores de superóxido (Mongkolsuk et al., 1998 e Ochsner et al., 2001). Por esta razão, este gene foi denominado ohr (organic hydroperoxide resistance gene). O objetivo desse trabalho foi caracterizar funcionalmente a proteína ohr de X. fastidiosa. Inicialmente, demonstramos que ohr possui atividade peroxidase dependente de tiól sendo que sua capacidade de reagir com hidroperóxidos é devida á presença de um par de cisteínas conservadas em seu sítio ativo. Também mostramos que ohr possui um enovelamento alfa/beta único, não observado nas estruturas de outras peroxidases dependentes de tiól como peroxirredoxinas e glutationa peroxidases. Análises do sítio ativo de ohr mostraram que seus prováveis substratos são moléculas hidrofóbicas e alongadas. Corroborando esta hipótese, demonstramos que enzimas lipoiladas, classicamente relacionadas com o metabolismo intermediário, interagem física e funcionalmente com ohr, enquanto que os sistemas tiorredoxina e glutationa, classicamente relacionados a tióis peroxidases, não sustentam a atividade peroxidásica de ohr. Este resultado representa a primeira descrição de uma peroxidase que é diretamente reduzida por grupos lipóicos de enzimas. Também fornecemos evidências que indicam que ohr atua na redução de hidroperóxidos derivados de ácidos graxos insaturados. De fato, análise cinética de estado estacionário por bi substrato mostra que ohr decompõem hidroperóxidos orgânicos com alta eficiência (kcat/KM ~ 106M-1.s1) através de um mecanismo ping-pong, sendo aproximadamente dez mil vezes mais eficiente do que na presença de H2O2. Esses dados em conjunto mostram que ohr é central na resposta bacteriana contra o estresse induzido por hidroperóxidos orgânicos, mas não por H2O2 e define uma nova classe de enzimas antioxidantes com propriedade únicas: peroxidases dependentes de grupos lipóicos. Outro objetivo desse trabalho foi estudar a via de regulação gênica de ohr em Xylella fastidiosa. Na maioria dos organismos, ohr é regulada por uma proteína repressora denominada ohrR (Sukchawalit et al., 2001), mas em algumas bactérias foi descrito que a expressão de ohr era regulada positivamente por um fator sigma alternativo (&#963;E) de função extra citoplasmática (Gourion et al., 2008). Nossos resultados mostraram que ohr de X. fastidiosa não está sob controle de nenhuma dessas proteínas, sendo provavelmente expressa constitutivamente. Análises por northern blot não mostraram alterações nos níveis de ohr em células submetidas a estresse oxidativo ou etanólico. Esses resultados, ainda que preliminares, indicam que possivelmente o controle da expressão gênica de ohr em X. fastidiosa é distinto daqueles descritos até o momento na literatura para outras bactérias. / Xylella fastidiosa is a gram-negative bacterium, which colonizes the xylem from economically important plants, being responsible for several diseases such as Pierce disease (PD) in gravepines and citrus variegated clorosis (CVC). Plants, when infected by pathogens, are able to defend themselves through several mechanisms which include the generation of reactive oxygen species (ROS). Lipid hydroperoxides can be generated from the attack of ROS to the bacterial membrane or by the action of lipoxygenases. The alkyl hydroperoxide reductase system (AhpR) was initially characterized as the main responsible for the detoxification of organic hydroperoxides in bacteria. Recently, it was also characterized another gene in many pathogenic bacteria, whose deletion renders cells susceptibility to organic hydroperoxide treatments but not by H2O2 or by superoxide generators (Mongkolsuk et al., 1998 and Ochsner et al., 2001). For this reason, it was named ohr (organic hydroperoxide resistance gene). The goal of this work was to functionally characterize ohr, the product of ohr gene from Xylella fastidiosa. Initially, we demonstrated that ohr possesses Cys-based thiol-dependent peroxidase activity. Later, we showed that ohr possesses a unique alpha/beta fold not observed in the structures of other thiol peroxidases such as peroxiredoxins and glutathione peroxidases. Analyses of ohr active site showed that its likely substrates are elongated and hydrophobic molecules. Furthermore, we showed that lipoylated enzymes, classically related with the intermediary metabolism, interacts physically and functionally with ohr while classical thiol-dependent pathways, such as thioredoxin and glutathione, failed to support ohr activity. This finding represents the first evidence of a peroxidase that is directly reduced by lipoyl groups of enzymes. Also, we obtained evidences indicating that ohr acts in the detoxification of peroxides derived from unsaturated fatty acids. In fact, steady-state kinetics using bi-substrate analysis showed that ohr decomposes organic peroxides with high efficiency (kcat/KM ~ 106 M-1.s-1 through a ping-pong mechanism, at least ten thousand times more efficiently than hydrogen peroxide (H2O2). All these results together shows that ohr is central in the response of bacteria to the stress induced by organic hydroperoxides but not by H2O2 and defines a new class of antioxidant enzymes with unique properties such as lipoyl-dependent peroxidase activity. Another goal of this work was to study the regulation of ohr expression in Xylella fastidiosa. ohr expression is regulated in most bacteria by a repressor protein named ohrR (Sukchawalit et al., 2001) but, in some bacteria, ohr expression is positively regulated by an alternative sigma factor (&#963;E) with extracitoplasmatic function (Gourion et al., 2008). Our results showed that ohr from X. fastidiosa was not under the control of none of these regulators, probably being constitutively expressed. Through northern blot analysis, we did not observed any changes in ohr levels in cells submitted to oxidative or ethanolic stress. These results, indicates that ohr expression probably differs from that previously described on literature for other bacteria.

O chumbo (Pb) é um metal altamente tóxico no qual os sinais de intoxicação variam bastante ao considerar as diferenças interindividuais. Um dos principais mecanismos de toxicidade do Pb ocorre pela inibição da enzima ácido delta aminolevulínico desidratase (ALAD) no sistema hematopoiético. O Pb também desempenha um importante papel no desbalanço do estado redox, pois sabe-se que ele tem o potencial de aumentar a concentração de espécies reativas de oxigênio (EROS) e inibir enzimas antioxidantes, como por exemplo a glutationa peroxidase (GPx). No entanto, poucos estudos avaliaram estes parâmetros em população ocupacionalmente exposta brasileira. Assim, o presente estudo, objetiva estudar a correlação entre as concentrações de Pb no sangue (Pb-S) de trabalhadores de fábricas de bateria e as atividades das enzimas ALAD e GPx associados com os polimorfismos genéticos da ALAD e GPx. Para tal, foram utilizadas 278 amostras de sangue de trabalhadores expostos ao Pb. As determinações de Pb foram realizadas por espectrometria de massas com plasma acoplado indutivamente (ELAN DRCII Perkin- Elmer). As genotipagens dos polimorfismos genéticos da ALAD e da GPx foram realizadas pela Reação em Cadeia da Polimerase (PCR) em tempo real e as atividades das enzimas ALAD e GPx foram determinadas no sangue por espectrofotometria de UV/VIS. A média da concentração de Pb-S foi de 22,8 ± 14,7 ?g/dL. Foram observadas correlações negativas entre Pb-S e atividade da ALAD (rs -0,24 p<0,01) e Pb-S e atividade de GPx (rs -0,27 p<0,05). Também foi verificada correlação negativa entre a porcentagem de inibição da ALAD e a atividade de GPx (rs -0,21 p<0,01). Em relação aos polimorfismos genéticos, não observamos associação entre os genótipos do ALAD (? -0,19 P>0,05) e GPx (genótipo CT: ? -1,37 P>0,05; genótipo TT: ? -8,37 P>0,05) e a concentração de Pb-S. Não foram observadas associações entre o polimorfismo rs1800668 e a atividade de GPx (genótipo CT: ? -0,016 P>0,05; genótipo TT: ? -0,004 P>0,05). No entanto, foi constatada uma associação entre o genótipo ALAD 1-1 do polimorfismo rs1800435 e a atividade da enzima ALAD (? 3,5 P<0,05). Neste sentido, os resultados deste estudo mostram que o genótipo ALAD 1-1 do polimorfismo rs1800435 do gene ALAD está associado a uma maior atividade da enzima ALAD nos indivíduos expostos ao Pb. Além disso, o polimorfismo rs1800668, localizado no gene que codifica a enzima GPx, não modula a atividade desta enzima nos indivíduos expostos ao metal. Ambos os polimorfismos dos genes ALAD e GPx parecem não influenciar nas concentrações de Pb-S na população estudada. / Lead (Pb) is a highly toxic metal which signs of intoxication vary greatly when considering the interindividual differences. One of the main mechanism of toxicity of Pb is due to inhibition of the enzyme acid delta aminolevulinic dehydratase (ALAD) in the hematopoietic system. Pb also plays an important role in the redox state of unbalance, since it is known that it has the potential of increasing the concentrations of reactive oxygen species (ROS) and inhibit antioxidant enzymes such as glutathione peroxidase (GPx). However, few studies have evaluated these parameters in Brazilian population occupationally exposed. Thus, the present study aims to study the correlation between the concentrations of Pb in the blood (B-Pb) of workers of battery factories and the activities of ALAD and GPx enzymes associated with genetic polymorphisms in ALAD and GPx. Then, blood samples of 278 workers exposed to Pb were collected. Pb was measured by Inductively Coupled Plasma-Mass Spectrometry (Perkin-Elmer ELAN DRCII). The genotyping of genetic polymorphisms of ALAD and GPx was performed by Real-Time Polymerase Chain Reaction (PCR) and activity of ALAD and GPx enzymes in the blood was determined by spectrophotometry UV / VIS. The mean concentration of B-Pb was 22.81 ± 14.73 mg / dL. Negative correlation was found between B-Pb and ALAD activity (rs -0.24 p < 0.01) and B-Pb and GPx activity (rs -0.27 p < 0.05). There was also a negative correlation between the percentage of inhibition of ALAD and GPx activity (rs -0.21 p < 0.01). Considering the genetic polymorphisms, no association between ALAD genotypes (? -0.19 P < 0.05) and GPx (CT genotype: ? -1.37 P > 0.05, TT genotype : ? -8.37 P > 0.05 ) and the concentration of B-Pb. No association between rs1800668 polymorphism and GPx activity (CT genotype: ? -0.016 P > 0.05, TT genotype : ? -0.004 P > 0.05). However an association was found between ALAD 1-1 rs1800435 genotype and ALAD enzyme activity (? 3.5 P < 0.05). Accordingly, the results of this study show that the ALAD 1-1 genotype of rs1800435 polymorphism in ALAD gene is associated with increased activity of ALAD enzyme in individuals exposed to Pb. Furthermore the rs1800668 polymorphism located in a gene encoding the enzyme GPx does not modulate the activity of this enzyme in individuals exposed to the metal. Both polymorphisms of ALAD and GPx genes seem to have no influence in the concentrations of B-Pb in the population studied.