A soluble enzyme system has been isolated from biotin-deficient rat liver acetone powder which catalyzes the synthesis of propionyl holocarboxylase from d-biotin and its apocarboxylase, in the presence of ATP and Mg++ ions. The enzyme system has been partially purified by (NH₄)₂SO₄ precipitation and resolved into two obligatory components by alumina C y gel fractionation. The gel supernatant fraction has been further purified by DEAE cellulose ion-exchange chromatography. Since the active component in the gel supernatant fraction and the endogenous propionyl holocarboxylase have the same elution pattern when chromatographed on DEAE cellulose, it appears likely that the gel supernatant contains the propionyl apocarboxylase. The gel eluate most likely contains an enzyme which catalyzes the covalent bonding or d-biotin to propionyl apocarboxylase and other proteins. In order to measure the activity of the propionyl holocarboxylase synthesizing system, two assay procedures were used. One assay procedure employed the incorporation of biotin-1-c¹⁴ into protein as a measure or the activity of the enzyme system. The other assay was based upon the biotin and ATP dependent increase of propionyl carboxylase activity catalyzed by the enzyme system. Both procedures gave similar results. Propionyl holocarboxylase formation was found to be ATP specific since neither CTP, OTP, ITP, nor UTP could replace ATP. In addition, a mixture or all five nucleoside triphosphates was no more effective than ATP alone. Versene inhibited the reaction and MgCl₂ was able to reverse this inhibition, indicating a Mg++ ion requirement. The ability of various biotin derivatives to replace d-biotin in propionyl holocarboxylase formation was investigated. It was round that if either the valeric acid side chain is altered. as in homo- or nor-biotin, of if the sulfur atom is removed or substituted, as in desthiobiotin or oxybiotin, holocarboxylase formation did not occur. Similarly, neither of these derivatives inhibited the bonding of c¹⁴-biotin to protein. Biocytin has been eliminated as an intermediate in propionyl holocarboxylase formation. Hydroxylamine does not inhibit the reaction. nor is CoA required. These data would appear to eliminate the involvement of free carboxyl activated biotinyl intermediates in the formation of the holocarboxylase from its apocarboxylase and biotin. Although the evidence suggests a concerted mechanism for the reaction, mechanisms involving enzyme bound intermediates are not completely eliminated by these data. In order to determine the nature of the attachment of biotin to propionyl carboxylase, c¹⁴-biotin labeled propionyl carboxylase was prepared. The labeled carboxylase was enzymatically hydrolyzed and chromatographed on Whatman 3MM paper. The biocytin peak contained nearly 100% of the radioactivity recovered. This peak was eluted and rechromatographed by ion-exchange chromatography. The only radioactive component obtained by this procedure was biocytin. The data presented indicate that the propionyl holocarboxylase synthesizing system catalyzes the ATP dependent covalent bonding of d-biotin to the lysyl-(-amino groups or propionyl apocarboxylase. / Ph. D.

Orientador: José Paschoal Batistuti / Banca: Fernando Luis Fertonani / Banca: Valeria Monterio da Silva Eleutério / Resumo: O presente trabalho consiste no desenvolvimento de um método analítico para determinação de biotina, utilizando-se a potenciometria indireta. O objetivo é apresentar a validação de método analítico como um processo que estime a eficiência do método proposto na rotina do laboratório para garantia da qualidade total. É um método que envolve equipamento simples e pouco dispendioso como o potenciômetro, que possibilita medir com precisão o valor da concentração de biotina. O doseamento quantitativo de biotina baseia-se no estudo das reações oscilantes do analito perante as análises da titulação indireta utilizando potenciômetro automático. A concentração de biotina foi determinada com massa adicionada conhecida numa matriz que simula uma cápsula, contendo aerosil (1%), estearato de magnésio (1%), celulose microcristalina (20%), amido (40%) e lactose q.s.p. Os resultados mostraram um valor médio de biotina (massa adicionada de aproximadamente 25 mg) determinada de 99,4% com desvio-padrão de 0,0345. As condições experimentais como temperatura, vidraria e concentração dos reagentes foram otimizadas. Os parâmetros investigados no processo de validação para demonstrar o desempenho do método foram: especificidade, linearidade, intervalo, precisão tanto repetitividade, quanto intermediária, exatidão e robustez. O tratamento estatístico dos dados da validação do método analítico envolveu a determinação da média, do desvio padrão e do coeficiente de variação. Para obtenção da curva de calibração se fez necessária a determinação da equação da reta, regressão linear e coeficiente de correlação linear. Este método apresenta grande aplicabilidade em soluções turvas, fluorescentes, opacas ou coradas, ou quando não existem, ou não podem aplicar-se indicadores visuais apropriados. Há possibilidade de determinação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work shows the development of an analytical method for the determination of biotin by using indirect potentiometry. The main goal is to present the validation of analytical methods as a process to estimate the efficiency of the proposed methodology in the laboratory routine for the guaranty of total quality. This method involves simple and low cost devices as the potentiometer, which allows the precise measurement of biotin concentration. The quantitative dosing of biotin is based on the study of the oscillating chemical reactions with the analyte by performing the analysis of the indirect titration with an automatic potentiometer. Biotin concentration was determined by adding a known mass to a matrix that simulates a capsule containing aerosol (1%), estearato de magnésio (1%), celulose microcristalina (20%), amido (40%) e lactose q.s.p. The results showed an average value for the determination of biotin (added mass was ca. 25 mg) of 99.4% with a standard deviation of 0.0345. The experimental conditions as temperature, glassware, and concentration of the chemicals were optimized. The investigated parameters of the validation procedure to demonstrate the performance of the method were: specificity, linearity, interval, precision (repeatability and intermediate), exactness and robustness. The statistical treatment of the data for the validation of the analytical method involved the determination of the average value, standard deviation, and the variation coefficient. In order to obtain the calibration curve, the line's equation, the linear regression and the coefficient of linear correlation were determined. This method shows great applicability for turbid, fluorescent, opaque or color solutions, or if an appropriate visual indicator is not available or cannot be applied. The method also shows the possibility for the determination of a sequence of equivalence... (Complete abstract click electronic access below) / Mestre

No description available.

<p>Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.</p><p>The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.</p><p>Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected.</p><p>The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.</p>

Biotinylated and non-biotinylated copolymers of ethylene oxide (EO) and 2-(diethylamino)ethyl methacrylate (DEAEMA) were synthesized by the atom transfer radical polymerization technique (ATRP). The chemical compositions of the copolymers as determined by NMR are represented by PEO₁₁₃PDEAEMA₇₀ and biotin-PEO₁₀₄PDEAEMA₉₃ respectively. The aggregation behavior of these polymers in aqueous solutions at different pHs and ionic strengths was studied using a combination of potentiometric titration, dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM). Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers form micelles at high pH with hydrodynamic radii (Rh) of about 19 and 23 nm, respectively. At low pH, the copolymers are dispersed as unimers in solution with Rh of about 6-7 nm. However, at a physiological salt concentration (cs) of about 0.16M NaCl and a pH of 7-8, the copolymers form large loosely packed Guassian chains, which were not present at the low cs of 0.001M NaCl. The critical micelle concentrations (CMC) and the cytotoxicity of the copolymers were investigated to determine a suitable polymer concentration range for future biological applications. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers possess identical CMC values of about 0.0023 mg/g, while the cytotoxicity test indicated that the copolymers are not toxic up to 0.05mg/g (> 83% cell survival at this concentration). / Singapore-MIT Alliance (SMA)

Investigations detailed in this thesis constitue a part of continuing programme of research work undertaken in this laboratory on vitamin binding proteins. Avidin from the chicken egg white, streptavidin &om the bacterium Streptromyces avidin and biotin binding proteins (BBP-I and BBP-11) from chicken egg yolk constitute a family of proteins that bind the vitamin biotin with extremely high affinities. The yolk BBPs are involved in the deposition of the vitamin in the developing oocyte in chicks whereas an antimicrobial function has been attributkl to avidin.. The fact that all these proteins bind the vitamin in the same manner, unlike biotin-dependent enzymes, indicates that the structural features involved in ligand binding could be similar, if not identical in these proteins. To delineate the basis of putative structural similarity among these proteins, studies were carried out using antibodies as the immunological probes. Avidin, a homotetremer glycoprotein, with a subunit Mr of 17,000 has been purified to homogeneity from chicken egg white using a novel procedure involving ammonium sulphate fractionation, ethanol precipitation and S-Sepharose column chromatography. Despite their lesser abundance in chicken egg yolk associated with a large amount of interfering lipids during the purification, both BBP-I (monomer and shown to be precursor for BBP-11) and BBP-I1 (tetramer) have been purified to homogeneity by employing a common method using butanol extraction to remove the lipids, DEAE-Sephacel column chromatography, biotin-AH-Sepharose affinity chromatography and fast performance liquid chrometography (FPLC) system. The purity of all these proteins was confirmed by SDS-PAGE analysis.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Title from first page of PDF file (viewed March 8, 2010). Includes bibliographical references. Issued in print and online. Available via ProQuest Digital Dissertations.

No description available.

Biotin is a water soluble vitamin that is mainly used as a cofactor in carboxylation reactions by a class of enzyme known as biotin-dependent carboxylases. In order to act as a cofactor, the biotin molecule has to be covalently attached to a lysine residue by an enzyme called holocarboxylase synthetase (HCS). Inherited deficiency of the biotin-dependent propionyl-CoA carboxylase (PCC) results in the inborn error of metabolism propionic acidemia. Mutations in either the alpha (PCCA gene) or beta (PCCB gene) subunit of the enzyme have been shown to cause propionic acidemia. Mutation analysis of the PCCB gene have revealed several mutations. However, few PCCalpha mutations have been described. The first goal of this thesis was to determine the molecular etiology of alpha subunit deficiency at the mRNA as well as at the protein level. I found that most mutations destabilized either the mRNA or the protein. Two other mutations were found to affect the biotinylation of PCCalpha, defining residues important for the folding of the domain or for interaction with HCS. The second part of my thesis was to study in more details the interactions between HCS and the biotinylation domain of PCCalpha, represented by the last 67 amino acids of the subunit (p-67). I expressed and purified p-67 from Pichia pastoris. I compared p-67 with the E. coli biotinylation domain (BCCP87) as substrates for the E. coli orthologous enzyme BirA, using steady-state as well as stopped-flow kinetics. I noticed some differences between these two substrates and how it might relate to the biotinylation reaction. I generated N-terminal and C-terminal deletions of HCS and I tested their activity in vivo and in vitro using purified susbtrates. I was able to map the minimal sequence requirement for HCS activity to the last 348 amino acids of the enzyme. I also found that some longer HCS were either almost or totally inactive or some that were active showed a differential activity towards the different susb

The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over