Catalase HPII of Escherichia coli is similar in sequence and structure to other catalases including the conservation of several residues on both the distal and proximal sides of the active center heme. The roles of many residues on the distal side of the heme have been well characterized. By contrast, very few residues on the proximal side of the heme or in the plane of the heme have been investigated. The primary goal of this thesis is to develop a better understanding of the role of the residues and structural features at the core of catalases and in the lateral access channel. The results demonstrate that a break in molecular symmetry does not have any functional significance. Replacing Ile274 with a Cys resulted in the heme being covalently linked to the protein through a Cys-vinyl bond which is hypersensitive to X-ray irradiation being largely degraded within seconds of exposure to the X-ray beam.

Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 224-238).

Th. doct.-ing.--Chim. minérale phys.--Saint-Étienne--École nationale supérieure des mines, 1976. N°: 6 C.I.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto. / Submitted by Maurílio Figueiredo (maurilioafigueiredo@yahoo.com.br) on 2015-01-28T20:33:18Z No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_InfluênciaSuplementaçãoCafeina.pdf: 25302484 bytes, checksum: 36134eb93fcec49c878f070d4b0c917a (MD5) / Approved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2015-01-30T18:26:56Z (GMT) No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_InfluênciaSuplementaçãoCafeina.pdf: 25302484 bytes, checksum: 36134eb93fcec49c878f070d4b0c917a (MD5) / Made available in DSpace on 2015-01-30T18:26:56Z (GMT). No. of bitstreams: 2 license_rdf: 22190 bytes, checksum: 19e8a2b57ef43c09f4d7071d2153c97d (MD5) TESE_InfluênciaSuplementaçãoCafeina.pdf: 25302484 bytes, checksum: 36134eb93fcec49c878f070d4b0c917a (MD5) Previous issue date: 2012 / Devido à utilização da cafeína como suplemento por atletas; sua relação com o estresse oxidativo gerado pelo exercício físico; e do alto consumo de café no Brasil, de sua importância como fonte de compostos bioativos na dieta, o objetivo deste trabalho foi avaliar se a suplementação de cafeína influencia na performance física de ratas submetidas ao treinamento físico e sua relação com o estresse oxidativo. Para isso, foram realizados 2 experimentos, sendo o primeiro realizado com 40 ratas da linhagem Fischer, divididas em 4 grupos com 10 animais cada. Os grupos foram: C-controle, CC-controle+cafeína, T-treinado, TC-treinado+cafeína. Os animais dos grupos C e T receberam dieta AIN-93M e os animais dos grupos CC e TC, dieta AIN-93M + cafeína (12 mg/Kg/dia). No segundo experimento, foram utilizadas 28 ratas, sendo divididas nos mesmos 4 grupos com 7 animais cada. A dose de cafeína utilizada foi de 15 mg/Kg/dia. Nos 2 experimentos, os animais praticaram natação 5 dias/semana, 30 minutos/dia, durante 7 semanas e com aumento progressivo da carga, de 1 a 5% do peso corporal. Para os animais do 2º experimento, foram realizados dois testes de exaustão, sem carga. O primeiro após 30 dias de dieta suplementada ou não com cafeína e o segundo, no final do experimento. Esses animais foram submetidos ao teste do lactato, onde nadaram com carga de 7,5% do peso corporal. A ingestão alimentar, o ganho de peso foram monitorados semanalmente e após 7 semanas, os animais foram sacrificados, o sangue e os órgãos foram coletados para dosagens bioquímicas e de estresse oxidativo. Os resultados do primeiro experimento mostraram que os efeitos da cafeína foram: aumento da ingestão alimentar; elevação do peso relativo do fígado, do músculo gastrocnêmio, da gordura abdominal e dos níveis de PON (paraoxonase). Os resultados do segundo experimento mostraram que a suplementação da dieta com cafeína não aumentou o tempo de natação e a concentração de lactato sanguíneo não chegou à sua estabilização. Quanto aos seus efeitos, a cafeína foi responsável por elevar o ganho de peso; aumentar a massa muscular; elevar os níveis de TBARS (Substâncias Reativas ao Ácido Tiobarbitúrico) no músculo gastrocnêmio, a catalase no músculo sóleo e a proteína carbonilada no fígado. Quando associada ao grupo controle, a cafeína reduziu a proteína carbonilada no músculo gastrocnêmio e reduziu a atividade da catalase no fígado e rins quando associada ao grupo treinado. Além disso, quando associada ao grupo treinado elevou as concentrações de creatinina e glutationa e a atividade da catalase no coração. A adição de 12 mg de cafeína/kg na dieta de ratas Fischer mostrou um efeito positivo sobre a atividade da PON (paraoxonase) e a dose de 15 mg/kg apresentou uma ação tecido específica para as defesas antioxidantes. __________________________________________________________________________________________ / ABSTRACT: Caffeine has been used as a supplement by athletes and has association with oxidative stress that is generated by exercise, and due the high consumption of coffee in Brazil and its importance as a source of bioactive compounds in the diet, the aim of this study was to evaluate if caffeine supplementation influences on physical performance of rats subjected to physical training and its association with oxidative stress. Two experiments were carried out. The first one used 40 female Fischer rats, divided into 4 groups of 10 animals each. The groups were: C-control, CC-control+caffeine, T-trained, TC-trained+caffeine. The animals in groups C and T were fed with AIN-93M diet and animals of CC and CT groups with AIN-93M+caffeine (12 mg/kg/day) diet. In the second experiment, 28 female Fischer rats were used, divided in the same groups of seven animals each. The caffeine level used in the diet was 15 mg/kg/day. In the two experiments, the animals practiced swimming 5 days/week, 30 min/day during 7 weeks and the load was progressively increasing from 1 to 5% of body weight. In the animals of the second experiment, two exhaustion tests without load were performed. The first test was in 30 days of diet supplemented or not with caffeine, and the second at the end of the experiment. These animals were submitted to the lactate testing, they swam with a load of 7.5% of body weight. Food intake, weight gain was monitored weekly and after seven weeks, the animals were sacrificed, blood and organs were collected for biochemical and oxidative stress assays. The results of the first experiment showed that the effects of caffeine were: increasing food intake, raising the relative weights of liver, gastrocnemius muscle, abdominal fat and levels of PON. The results of the second experiment showed that supplementing the caffeine in the diet did not increase the swimming time and blood lactate concentration did not reach its stabilization. As to its effects, caffeine was responsible for raising the weight gain, increase muscle mass, increase levels of TBARS in the gastrocnemius muscle, catalase in the soleus muscle and protein carbonyl in the liver. When associated to the control group, caffeine reduced the protein carbonyl in gastrocnemius muscle and as well as the activity of catalase in the liver and kidneys when associated with the trained group. Moreover, when associated with the trained group increased creatinine concentrations and glutathione and catalase activity in the heart. The addition of 12 mg caffeine/kg in the diet of female Fischer rats showed a positive effect on the activity of PON and addition of 15 mg/kg showed an response tissue-specific in the antioxidant defenses.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química. / Made available in DSpace on 2012-10-20T00:59:13Z (GMT). No. of bitstreams: 1 184979.pdf: 642247 bytes, checksum: 0f983b65abb404168542c1b891ba1927 (MD5) / Síntese e caracterização de um novo ligante não-simétrico binucleante heptadentado, bem como de seu complexo tetranuclear de manganês. O ligante foi caracterizado por espectroscopias eletrônica, vibracional e de ressonância magnética nuclear. O complexo foi caracterizado por difratometria de raios-X, análise elementar, espectroscopias eletrônica, vibracional e paramagnética eletrônica, eletroquímica e espectro-eletroquímica. Realizou-se um estudo de cinética química para investigar o mecanismo de decomposição do peróxido de hidrogênio catalisada pelo presente complexo.

No / Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme. / The full-text of this article will be released for public view at the end of the publisher embargo on 19 Apr 2022.

No description available.

Macrocycle (H2L1) was prepared by a Schiff base condensation reaction of 2,6-diformylpyridine and 1,3-diamino-2-propanol in the presence of Ba(II) as template ion. Seven-coordinate Mn(II) complexes were prepared by transmetallation reactions of the initial [Ba(H2L1)(μ1,2-ClO4)]2(ClO4)2 complex. Two mononuclear, ring-contracted complexes were obtained when methanol or ethanol were used as solvents in transmetallation reactions. For both complexes, X-ray analysis showed that the H2L1 macrocycle undergoes a ring-contraction via addition of methanol or ethanol across one imine bond, followed by a nucleophilic addition of the secondary amine across an adjacent imine bond resulting in a six-membered, hexahydropyrimidine ring sitting in a chair conformation. The ring-contraction process reduces the size of the cavity in the macrocycle to accommodate one Mn(II) ion in the macrocycle. The macrocyclic tetraimine ligand (H2L1) gave access to the polynuclear, ring-expanded assemblies, [Mn4(H2L*)Cl4][MnCl4] and [Mn4(H2L*)(N3)4](ClO4)2, when acetonitrile was used as a solvent. The macrocycle (H2L1) undergoes rearrangement from a 20-membered to a 40-membered tetranuclear Mn(II) complex. Manganese complexes of acyclic ligands, derived from 2,6-diformylpyridine and several aminoalcohols and aminophenols, were prepared and structurally characterised by X-ray crystallography. Most of the complexes are seven-coordinate with approximate pentagonal bipyramidal geometry, however, some five, six and seven-coordinate complexes were identified. Asymmetric and symmetric tripodal Schiff base ligands and their manganese complexes were also prepared and characterised. Additionally, N-alkylated benzimidazole 2,6-bis(1-butyl-1H-benzo[d]imidazol-2-yl)pyridine and its Mn(II) complexes were prepared and characterised. The potential application of the complexes has been tested in two main areas: (a) as new catalase mimics and (b) as new superoxide dismutase (SOD) mimics. The trinuclear, acyclic complex, [Mn3(L9)2(OAc)2(MeOH)2] 2MeOH, derived from 2,6-diformylpyridine and 2-aminophenol, was found to be the most efficient catalase mimic of the tested complexes with approximately 500 molecules of H2O2 broken down per second for each complex during the fastest rate of activity. Catalase testing showed that an increase of the arm size of the tripodal complexes produced an increase in activity overall for the complexes. Most of the complexes tested for catalase activity showed an induction period prior to the activity being observed. This may be due to a rearrangement occurring before catalase activity is observed. The tripodal complex, [Mn(L18)](ClO4)2 is the only complex to show a catalase activity without added base, but with a long induction period. The results that are presented indicate that the axial ligands have an effect on both the rate of catalase activity and the observed induction period. The SOD results indicated that the complex, [Mn(H2L6)Cl(H2O)]Cl H2O, derived from 2,6-diformylpyridine and 1 aminopropan-2-ol, shows the highest SOD activity amongst the complexes prepared, with a rate of 2.05x106 M-1s-1 and the IC50 value of 0.78 μM. Most of the complexes showed SOD activity with a rate around 105-106 M-1s-1. The SOD results showed that the axial ligands have an effect on SOD activity; strongly bound ligands such as thiocyanate and azide generally result in lower SOD activity. Most of the complexes showed both SOD and catalase activity. Ring-contracted complexes, [Mn(H3L2)(NCS)2] and [Mn(H3L3)(NCS)2], show high rates of superoxide dismutase activity but possess limited catalase activity.

Neste trabalho, propomos o estudo dos mecanismos de adsorção de um novo material adsorvente capaz de extrair íons metálicos, obtido através da modificação química de nanotubos de carbono (CNT) com o emprego de agente oxidante ácido, seguido do revestimento com uma camada externa de albumina de soro bovino (BSA), resultando em nanotubos de carbono de acesso restrito revestidos com BSA (RACNT-BSA). Os materiais foram caracterizados por: análise termogravimétrica (TG/DTG) a fim de avaliar a estabilidade térmica, espectroscopia no infravermelho (IR) para confirmação dos grupos funcionais envolvidos nos processos oxidação dos CNT e recobrimento dos mesmos com BSA, análise elementar (CHN), microscopia eletrônica de varredura (MEV) para avaliar a morfologia e difração de raio-X. Para facilitar a compreensão dos mecanismos de interações entre os RACNT-BSA com BSA em solução e com íons metálicos, determinaram-se os pontos isoelétricos de todos os materiais. Para o estudo dos possíveis mecanismos de interação entre os RACNT-BSA e proteínas em solução, percolou-se soluções de BSA com concentração 3 μg. mL-¹ em tampão fosfato com pH no intervalo de 2,2 a 7,0 nos RACNT-BSA. Dessa maneira, verificou-se uma relação entre o processo adsortivo nos RACNT-BSA e o pI (ponto isoelétrico) das proteínas, ou seja, propomos um mecanismo para o funcionamento dos RACNT-BSA dependente do pH da solução e das proteínas a serem percoladas. Quando uma solução de proteínas é percolada através de um cartucho de SPE contendo RACNT-BSA e o pH da solução é maior do que o ponto isoelétrico das proteínas, ambas as proteínas solubilizadas e a camada de BSA são ionizadas negativamente. Assim, uma repulsão eletrostática impede a interação entre as proteínas da amostra e a superfície do RACNT-BSA. Verificou-se que os íons metálicos podem ser adsorvidos nos RACNT-BSA, porém os processos adsortivos são afetados quanto a variação do pH da solução e a escolha do tampão adequado. Também se estudou a extração dos íons Fe das estruturas das proteínas da catalase. Quanto à aplicação para a catalase, verificamos que ao mesmo tempo em que não há adsorção desta proteína nos RACNT-BSA, os íons Fe, ligados covalentemente à ela, são atraídos e adsorvidos nos RACNT-BSA, sendo necessário, porém a desnaturação proteíca. Isto nos mostrou que o material RACNT-BSA se mostrou promissor para a formação de apoproteínas. / In this work, we proposed to study the adsorption mechanisms of a new adsorbent material capable of extracting metal ions, obtained by chemical modification of carbon nanotubes (CNT) with the use of oxidizing acidic agent, followed by coating with an outer layer of bovine serum albumin (BSA), resulting in restricted access of carbon nanotubes coated with BSA (RACNT-BSA). The materials were characterized by thermogravimetric analysis (TG) in order to evaluate thermal stability, infrared spectroscopy (IR) to confirm the functional groups involved in the oxidation processes of CNT and covering the same with BSA, elemental analysis (CHN), scanning electron microscopy (SEM) to evaluate the morphology and powder X-ray diffraction. To facilitate the understanding of the interaction mechanisms among RACNT-BSA and BSA in solution and metal ions, the isoelectric points (Ip) (isoelectric point) of all materials were determined. To study the possible interaction mechanisms between RACNT-BSA and solubilized proteins, the BSA solutions (3 μg. mL-¹) in phosphate buffer with a pH in the range 2.2 to 7.0 were percolated through the RACNT-BSA. Thus, there is a relationship between the adsorptive process in the RACNT-BSA and the protein Ip’s, i.e., we proposed a working mechanism for RACNT-BSA dependent on the pH solution and on the type of percolated protein. When a protein solution is percolated through a SPE cartridge containing RACNT-BSA and the pH solution is higher than the protein Ip, both solubilized protein and BSA layer (from RACNT-BSA) are negatively ionized. Therefore, an electrostatic repulsion prevents the interaction between the solubilized proteins and the surface of RACNT-BSA. It was found that metal ions can be adsorbed on RACNT-BSA, but the adsorptive processes can be affected according the changing of pH solution, and the choice of the appropriate buffer. The application to catalase protein, we found that while there is no adsorption of this protein on RACNT-BSA, the Fe ions, covalently linked to it, are attracted and adsorbed on RACNT-BSA, requiring, however, the denaturation of protein. These results showed us that the RACNT-BSA material is promising for the apoprotein’s formation. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Lai Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 93-100). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / ABBREVIATION --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Catalases --- p.2 / Chapter 1.2 --- Classification of catalases --- p.3 / Chapter 1.2.1 --- Catalase peroxidase (HPI) --- p.3 / Chapter 1.2.2 --- Monofunctional catalases (HPII) --- p.6 / Chapter 1.2.2.1 --- NADPH in catalases --- p.9 / Chapter 1.2.3 --- Mn-catalases --- p.11 / Chapter 1.3 --- Sources and cytotoxic effects of hydrogen peroxide --- p.13 / Chapter 1.4 --- The Chironomidae --- p.14 / Chapter 1.4.1 --- Life cycle of Chironomidae --- p.14 / Chapter 1.4.2 --- Bloodworms --- p.18 / Chapter 1.4.3 --- Sources of bloodworms --- p.19 / Chapter 1.5 --- Aim of the project --- p.22 / Chapter 1.6 --- Application of the project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Protein determination --- p.25 / Chapter 2.2 --- In vitro activity assays --- p.27 / Chapter 2.2.1 --- Catalase activity assay --- p.27 / Chapter 2.2.2 --- Peroxidase activity assay --- p.27 / Chapter 2.3 --- Screening of catalase in different subcellular fractions --- p.28 / Chapter 2.3.1 --- Preparation of mitochondrial fractions --- p.28 / Chapter 2.3.2 --- Preparation of microsomal fractions --- p.29 / Chapter 2.3.3 --- Preparation of cytosolic fractions --- p.29 / Chapter 2.3.4 --- Preparation of post-mitochondrial fractions --- p.29 / Chapter 2.4 --- Purification of post-mitochondrial catalase --- p.29 / Chapter 2.4.1 --- Preparation of post-mitochondrial fractions --- p.30 / Chapter 2.4.2 --- Ethanol-chloroform precipitation --- p.30 / Chapter 2.4.3 --- Affinity chromatography --- p.30 / Chapter 2.4.4 --- Cation exchange chromatography --- p.31 / Chapter 2.5 --- Molecular mass determination --- p.34 / Chapter 2.6 --- Isoelectric focusing --- p.39 / Chapter 2.7 --- Kinetic studies of the purified enzyme --- p.42 / Chapter 2.7.1 --- Optimal pH --- p.42 / Chapter 2.7.2 --- Thermal stability --- p.42 / Chapter 2.7.3 --- Km and Vmax --- p.42 / Chapter 2.7.4 --- Inhibition studies --- p.43 / Chapter 2.7.4.1 --- "3-amino-1,2,4-triazole" --- p.43 / Chapter 2.7.4.2 --- Potassium cyanide and sodium azide --- p.43 / Chapter 2.8 --- Spectroscopic analysis --- p.44 / Chapter 2.8.1 --- Native enzyme --- p.44 / Chapter 2.8.2 --- Denatured enzyme --- p.44 / Chapter 2.8.3 --- Determination of pyridine hemochrome --- p.44 / Chapter 2.9 --- N-terminal amino acid sequence analysis for blotted protein --- p.45 / Chapter 2.9.1 --- Semi-dry electroblotting --- p.45 / Chapter 2.9.2 --- Protein staining on PVDF membrane --- p.46 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.46 / Chapter 2.9.4 --- N-terminal deblocking of protein bound on PVDF membrane… --- p.47 / Chapter 2.9.5 --- BLAST® search --- p.48 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Catalase in different sub-cellular fractions --- p.50 / Chapter 3.2 --- Purification of post-mitochondrial catalase --- p.51 / Chapter 3.2.1 --- Ethanol-chloroform precipitation --- p.51 / Chapter 3.2.2 --- Affinity chromatography --- p.51 / Chapter 3.2.3 --- Cation exchange chromatography --- p.52 / Chapter 3.3 --- Determination of molecular mass --- p.57 / Chapter 3.4 --- Determination of isoelectric point --- p.57 / Chapter 3.5 --- Kinetic studies of the catalase --- p.62 / Chapter 3.5.1 --- Optimal pH --- p.62 / Chapter 3.5.2 --- Thermal stability --- p.62 / Chapter 3.5.3 --- Km and Vmax --- p.65 / Chapter 3.5.4 --- Inhibition studies --- p.65 / Chapter 3.5.4.1 --- "3-amino-1,2,4-triazole" --- p.65 / Chapter 3.5.4.2 --- Potassium cyanide and sodium azide --- p.65 / Chapter 3.5.5 --- Catalase peroxidase activity --- p.66 / Chapter 3.6 --- Spectroscopic analysis --- p.73 / Chapter 3.6.1 --- Native enzyme --- p.73 / Chapter 3.6.2 --- Denatured enzyme --- p.73 / Chapter 3.6.2.1 --- Potassium cyanide --- p.73 / Chapter 3.6.2.2 --- Sodium azide --- p.73 / Chapter 3.6.3 --- Pyridine hemochrome characterization --- p.73 / Chapter 3.7 --- N-terminal amino acid sequence analysis --- p.79 / Chapter CHAPTER 4 --- DISCUSSION --- p.81 / Chapter 4.1 --- Subcellular locations of catalase in bloodworms --- p.82 / Chapter 4.2 --- Purification of post-mitochondrial catalase --- p.82 / Chapter 4.3 --- Physical properties of the purified enzyme --- p.84 / Chapter 4.3.1 --- Native and subunit molecular mass --- p.84 / Chapter 4.3.2 --- Isoelectric point --- p.85 / Chapter 4.4 --- Kinetic properties of the purified enzyme --- p.85 / Chapter 4.4.1 --- Optimal pH --- p.85 / Chapter 4.4.2 --- Thermal stability --- p.85 / Chapter 4.4.3 --- Km and Vmax --- p.87 / Chapter 4.4.4 --- Inhibition studies --- p.87 / Chapter 4.4.5 --- Catalase peroxidase activity --- p.87 / Chapter 4.5 --- Spectroscopic analysis --- p.88 / Chapter 4.5.1 --- Native and denatured enzyme --- p.88 / Chapter 4.5.2 --- Pyridine hemochrome characterization --- p.88 / Chapter 4.6 --- N-terminal amino acid analysis --- p.89 / Chapter 4.7 --- Conclusions --- p.89 / REFERENCES --- p.93