Kwok Leung Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-170). / Acknowledgments --- p.i / Abstract --- p.ii -v / Abbreviations --- p.vi-vii / Chapter Chapter 1 : --- General Introduction --- p.1 / Chapter Chapter 2 : --- Development of Polyclonal Antibodies Against 6-B-hydroxycortisol （6-B-OHC) And Its Applications / Chapter 2.1 : --- Introduction --- p.40 / Chapter 2.2 : --- Materials and methods --- p.43 / Chapter 2.3: --- Results --- p.55 / Chapter 2.4: --- Discussion --- p.73 / Chapter Chapter 3 : --- Development of Monoclonal Antibody-Based ELISA Against 6-B-hydroxycortisol (6-B-OHC) And Its Applications / Chapter 3.1 : --- Introduction --- p.76 / Chapter 3.2 : --- Materials and methods --- p.89 / Chapter 3.3: --- Results --- p.108 / Chapter 3.4: --- Discussion --- p.135 / Chapter Chapter 4 --- : General Conclusion --- p.141 / References --- p.149

Thesis. 1976. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Microfiche copy available in Archives and Science. / Vita. / Bibliography: leaves 231-249. / Ph.D.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-26Bitstream added on 2014-06-13T20:21:35Z : No. of bitstreams: 1 santos_mc_dr_araiq.pdf: 1124332 bytes, checksum: 1e349d4a6fad699f76e186f17e5de53c (MD5) / Outros / As peroxidases são hemeproteínas que catalisam a oxidação de vários xenobióticos na presença de peróxido. A soja é um dos principais produtos de exportação do Brasil e sua manufatura gera subprodutos em grande escala. Um destes, a casca, é uma fonte rica em peroxidase (SBP). Para a otimização da extração da SBP de casca de soja, e condições de ensaio, foi utilizado um planejamento fatorial com metodologia de superfície de resposta, resultando em 128 experimentos. Os dados definiram o meio reacional da SBP: pH 4,5 (transgênicas) e pH 7,0 (tradicionais); temperatura 500C; tratamento do tegumento com obtenção do pó cetonico; NaCl 0,1 mol/L; volume de amostra da enzima 330mL, guaiacol 30mmol/L, H2O2 46 mmol/L, em tampão 50mmol/L. Após, iniciou-se os estudos da SBP de diferentes cultivares: convencionais (BRS 258, BRS 260, BRS 262) e transgênicas (BRS 255 RR, BRS Charrua RR e BRS Pampa RR) de cascas de soja fornecidas pela EMBRAPA, visando estudo comparativo sobre o conteúdo de peroxidase (SBP), e posterior seleção para isolamento da enzima e estudos de seus parâmetros cinéticos. Para isso, a amostra, em pó cetônico, de cada cultivar foi ressuspensa em tampão extrator, a proteína precipitada com sulfato de amônio 70%, o precipitado ressuspenso em tampão extrator e eluído em coluna Sephadex G-25, para dessanilização. Nas frações (eluatos) foram realizados os spots test para identificação de SBP. As cultivares BRS 258, BRS 262, BRS 260 e BRS 255 RR foram selecionadas com maior conteúdo de enzima SBP. Assim, volumes (5mL) de tais cultivares foram eluídos em coluna Sephadex G-100 para purificação parcial e determinação da massa molecular. Com as frações (eluatos) obteve-se “pool” de enzima, onde se efetuou os estudos cinéticos: efeito de cátions mono e divalentes, determinação dos parâmetros cinéticos - kM, vmax, pH ótimo, temperatura ótima... / The peroxidases are hemeproteins that catalyze the oxidation of various xenobiotics in the presence of peroxide. Soybean is a major export products from Brazil and its manufacture creates byproducts in large scale. One of these, the bark is a rich source of peroxidase (SBP). To optimize the extraction of the SBP of soybean seed coat, and test conditions, we used a factorial planning with response surface methodology, resulting in 128 experiments. The data defined the reaction medium SBP pH 4.5 (transgenic) and pH 7.0 (traditional), temperature 500C; treatment of sample to obtain the “acetone powder”, NaCl 0.1 mol / L, sample volume of the enzyme 330 mL, guaiacol 30 mmol / L, H2O2 46 mmol / L in buffer 50mmol / L. After he began the studies of SBP from different cultivars: Conventional (BRS 258, BRS 260, BRS 262) and transgenic (BRS 255 RR, BRS and BRS Pampa RR) soybean seeds coat supplied by EMBRAPA, doing comparative study on the content of peroxidase (SBP), and subsequent selection to isolate the enzyme and studies of its kinetic parameters. For this, the sample in “acetone powder” of each cultivar was re-suspended in extraction buffer, the protein precipitated with ammonium sulfate 70%, the precipitate resuspended in extraction buffer and eluted in Sephadex G-25 column, for desalination. In the fractions (eluates) were performed to identify test spots SBP. BRS 258, BRS 262, BRS 260 and BRS 255 RR were selected with the highest content of enzyme SBP. Thus, volumes (5mL) of these cultivars were eluted in Sephadex G-100 column for partial purification and determination of molecular weight. With the fractions (eluate) obtained a pool of enzyme, which has made the kinetic studies: effects of mono-and divalent cations, determination of kinetic parameters - kM, vmax, optimum pH, optimum temperature. The data showed: i) optimum pH 4.5, ii) the optimum temperature, BRS 260... (Complete abstract click electronic access below)

The Classical Model describing the effects of temperature on enzyme activity consists of two processes: the catalytic reaction defined by ΔG cat and irreversible inactivation defined by ΔG inact, this model however, does not account for the observed temperature- dependant behaviour of enzymes. The recent development of the Equilibrium Model is governed not only by ΔG cat and ΔG inact but also by two new intrinsic parameters ΔHeq and Teq, which describe the enthalpy and the temperature of the midpoint, respectively, of a active and reversibly inactive enzyme transition. Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological and environmental aspects of life to temperature in a way that has not been previously possible. The Equilibrium Model is therefore a more complete and accurate description of the effects of temperature on enzymes, it has provided new tools for describing and investigating enzyme thermal adaptation and possibly new biotechnological tools. The effects of the incorporating in the new Model of the parameters Teq and ΔH eq yield major differences from the Classical Model, with simulated data calculated according to the Equilibrium Model fitting well to experimental data and showing an initial rate temperature optimum that is independent of assay duration. Simulated data simulated according to the Classical Model can not be fitted to experimental data. All enzymes so far studied (gt30) display behaviour predicted by the Equilibrium Model. The research described here has set out to: experimentally test observations made by Eisenthal et al., on the basis of enzyme reactor data simulated according to the Equilibrium Model; to test the Equilibrium Model using an unusual (rapidly renaturable) enzyme, RNAase; and to test the proposed molecular basis of the Equilibrium Model by examining the effect of a change at the enzymes active site. The experimental results gathered here on the effect of time and temperature on enzyme reactor output confirm the predictions made by Eisenthal et al. (2006) and indicate that the Equilibrium Model can be a useful aid in predicting reactor performance. The Equilibrium Model depends upon the acquisition of data on the variation of the Vmax of an enzyme with time and temperature, and the non-ideal behaviour of RNase A made it impossible to collect such data for this enzyme, as a result the Equilibrium Model could not be applied. The disulfide bond within the active site cleft of A.k 1 protease was cleaved as a probe of the mechanism of the Equilibrium Model, which is proposed to arise from molecular changes at the enzymes active site. Support for the proposed mechanism was gained through the comparison of experimentally determined temperature dependence of the native and reduced forms of the enzyme and application of this data to the Equilibrium Model.

In recent years, the methyl erythritol phosphate (MEP) pathway to isoprenoids has been the subject of intensive research. The interest is because isoprenoids have important roles in many cellular processes essential for the survival of several pathogenic organisms, making the inhibition of this pathway an attractive target for the drug discovery. The second enzyme in the MEP pathway is 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). DXR is a promising target for the development of new antibiotics, antimalarials and herbicides. The overall objective of this research was a better understanding of DXR by using site-directed mutagenesis guided by crystal structure analysis and inhibition studies. One set of mutants was designed to expand the selectivity of DXR. An analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP or Me-DXP), that differs from DXP by having an ethyl ketone, rather than a methyl ketone, was reported to be a weak competitive inhibitor. Using the x-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified as a potential steric block to the use of this analog as a substrate. Four mutants of Synechocystis sp. PCC6803 DXR, named W204F, W204L, W204V and W204A, were prepared and characterized. The W204F mutant was found to utilize the analog Me-DXP as a substrate. The roles of amino acids residues shown to be in the DXR active site in the available E. coli crystal structures were also studied. Mutants at the positions Dl52, S153, E154, H155, M206 and E233, were prepared. The kinetic characterization of these mutants showed that the amino acid substitution, conservative or not, in these residues reduced the DXR catalytic activity, confirming that these are key amino acids responsible for the DXR catalytic efficiency. Inhibition studies of the E. coli DXR by fosmidomycin in the presence of Co²⁺, Mg²⁺ and Mn²⁺ showed that this inhibition is not dependent on a specific divalent cation. Inhibition of the Synechocystis sp. PCC6803 DXR by fosmidomycin and its hydroxamate and FR 900098 analogs was conducted showing that these compound are potent inhibitors of this enzyme. Fosmidomycin and FR900098 have inhibition constants in the low nM range. In addition the patterns of the progress curves for fosmidomycin, its hydroxamate analog and FR900098 were shown to be prototypical for slow, tight-binding inhibitors, as was seen for these inhibitors with the E. coli enzyme. / Graduation date: 2005

Graduation date: 1997

Graduation date: 1996

Graduation date: 1996

Graduation date: 1994

There are some 15,000 known organophosphorus chemicals. Some of these OP’s, including VX and paraoxon, demonstrate an acute neurotoxicity due to the inhibition of cholinergic enzymes. Organophosphorus chemical warfare agents and pesticide neurotoxins are subject to hydrolysis by OP degrading enzymes. To be useful as a bioremediation or anti-chemical warfare agent, the enzyme must be tailored for, and integrated into, a practical application platform. Several studies have established enzyme-based countermeasures, describing such diverse applications as decontaminating foams for surface remediation, encapsulating enzyme with liposome for in vivo therapy, enzyme attachments to surfaces for biosensors and development of a corn expression system for large-scale enzyme production. The goal of the research described here is to select, investigate and improve the operational potential of organophosphate-degrading enzymes including Organophosphorus Hydrolase (OPH, 3.1.8.1) and Organophosphorus Acid Anhydrolase (OPAA, 3.1.8.2). Using saturation kinetics, the catalytic efficiencies of these two major detoxification enzymes were characterized with substrates representing each class of OP neurotoxin, phosphotriester, phosphothioate and phosphofluoridate. OPH presents superior kinetic parameters with each OP class tested. Variants of OPH were created to increase the operational effectiveness of OP hydrolytic enzymes against phosphorothioates. An H254S/H257L mutation in the active site resulted in an improvement in the kinetics (kcat/KM) for the phosphorothioate, demeton-S. To screen potential vascular protection therapies, an in vitro protocol was developed to predict enzymatic effectiveness for protection of acetylcholinesterase from acute OP-inhibition. The protection abilities of the enzymes were directly related to their second order rate constants as inhibitory levels of OP are below the KM of the enzymes. Consideration of contaminant nature concentration and enzyme kinetic parameters, kcat and KM, is critical to understanding decontamination and effective use of enzyme technology. These technologies continue to develop and provide promising new decontamination tools for OP compounds.