Made available in DSpace on 2014-06-12T15:49:40Z (GMT). No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bromelina é o nome genérico dado ao grupo de enzimas proteolíticas encontrada nos vegetais da família Bromeliaceae, da qual o abacaxi é o mais conhecido e rico nesta protease, que tem grande importância na área de saúde e de alimentos. O objetivo deste trabalho foi determinar as propriedades físico-químicas e purificar a bromelina presente no Ananas comosus L. Merril (abacaxi). A Diálise seguida da precipitação com 80% de (NH 4 ) 2 SO 4 realizado no extrato do abacaxi proporciou uma maior recuperação de atividade e proteína produzindo um fator de purificação cerca de 3 vezes. O CE e o CED80% apresentaram valores de pH ótimos de 8,5 e 7,2, respectivamente. Entretanto a bromelina foi estável em todos os valores de pH testados (5,0-9,0) mostrando assim ser formada por diferentes frações proteolíticas. Em relação à temperatura ótima, o CE e o CED80% apresentaram valor de 50ºC e foram foram estáveis até temperaturas de 60 ºC, sendo totalmente desnaturada a 80ºC após 30 minutos. Os valores de K M e V max aparentes, calculados segundo modelo de Lineweaver-Burk, foram: para azocaseína - K M = 2,48 mg/ml e V max = 35,29 U/mL e para azoalbumina - K M = 2,94 mg/ml e V max = 11,11 U/mL, mostrando assim praticamente a mesma afinidade por ambos os substratos. Entretanto a eficiência catalítica foi maior para azocaseína que para azoalbumina (31,62 e 8,39 min -1 , respectivamente). Através da eletroforese em gel SDS-PAGE, o extrato bruto apresentou 4 bandas de proteínas com massas moleculares de 40, 29 e 27 kDa e uma inferior a 14 kDa. Através de zimograma as bandas de 29 e a abaixo de 14kDa apresentaram atividade caseinolítica. A Cromatografia de gel-filtração apresentou 2 picos, entretanto apenas o pico 2 com atividade proteolítica especifica de 116 U/mg de proteína e purificação de 25,1 vezes, o qual apresentou 2 bandas de 29 e 26 kDa em gel SDS-PAGE. Pico 2 este quando aplicado na cromatografia de troca aniônica, rendendo um maior pico (P1) com atividade proteolítica o qual apresentou banda única em SDS- PAGE de 29 kDa com atividade caseinolítica comprovada em zimograma

La concentració de proteïnes dels vins blancs és de l'ordre de miligrams per litre. Aquestes proteïnes presenten en el vi càrrega elèctrica positiva degut a que tenen un punt isoelèctric superior al pH del vi. Aquesta càrrega les fa reaccionar amb les substàncies carregades negativament en el vi, especialment polifenols i alguns clarificants addicionats. Aquestes proteïnes influeixen negativament en la qualitat dels vins degut a la tendència a precipitar i produir enterboliments però confereix al vi propietats positives pel que respecta a la estabilització de l'escuma en els vins escumosos i a la influència sobre la volatilitat de les molècules responsables de la aroma dels vins blancs, augmentant llur permanència. El treball realitzat ha consistit en la posta a punt d'un mètode de separació de les proteïnes dels vins per mitjà de tècniques de cromatografia en columna a baixes pressions, coneguda com FPLC. Aquest mètode te una gran precisió i repetibilitat. Així es poden separar les proteïnes del vi en una proteïna de 60 kDa i un nombre variable en una franja de 20 a 30 kDa, que presenten diferent càrrega elèctrica. La fracció de inferior massa molecular presenta un perfil de separació segons la carrega elèctrica diferent per las cinc varietats viníferes estudiades: chardonnay, pinot noir, macabeu, xarel.lo i parellada. S'observa una disminució de la concentració de proteïna durant el procés de vinificació que afecta a totes les fraccions separades; no obstant aquesta davallada és major a les fraccions de 20 a 30 kDa de més càrrega elèctrica positiva, el que s'explica per una més gran reactivitat en front als compostos amb carrega negativa en el vi. S'ha estudiat la influència del temps de contacte amb els baixos fins de fermentació sobre les diferents fraccions de proteïnes sense observar cap variació significativa pel fraccionament estudiat. Al llarg de la maduració s'ha detectat un increment de la concentració de proteïna a la fracció de baixa massa molecular, mentre que la fracció de 60 kDa es manté en concentracions similars en el període estudiat. D'aquesta fracció de 20 a 30 kDa s'ha constatat un increment més gran de les proteïnes amb menys càrrega elèctrica. Després d'analitzar el fraccionament de la fracció de 20 a 30 kDa per bescanvi catiònic de les cinc varietats viníferes durant dos anys s'han observat perfils característics per cada varietat vinífera que permeten classificar-les en funció de les fraccions obtingudes, el que fa pensar en una possible eina per la caracterització varietal. / Proteins are present in white wines in small amounts (a few milligrams per litre). These proteins have a positive electric charge because their isoelectric point is higher than the pH of the wine. This charge makes them react with the negatively-charged substances in the wine, particularly polyphenols or fining agents. These proteins have a negative effect on the wine quality due to their tendency to precipitate and produce cloudiness but they are also positive because they stabilize the foam in sparkling wines and increase the permanence of the molecules responsible for the aroma of white wines.The study consisted of establishing a method for separating proteins from wines using column chromatography techniques at low pressures known as FPLC. This method has high precision and repeatability. Thus the proteins in wine can be separated into a protein of 60 kDa and a variable number of proteins ranging from 20 to 30 kDa, which have different electrical charges. The fraction with the lowest molecular weight has a separation profile which depends on the electrical charge for the five grape varieties studied: Chardonnay, Pinot Noir, Macabeo, Xarel·lo and Parellada.The protein concentration decreases during the vinification process in all the separated fractions. However, this decrease is more pronounced in the fractions from 20 to 30 kDa which had larger positive charges because they react more to the negatively-charged compounds in the wine. The effect of the contact time between the low fermentation fines and the different protein fractions was studied but no significant variation in the fractioning studied was observed. Throughout the maturation the protein concentration was seen to increase in the low molecular weight fraction, while the concentration of the 60 kDa fraction hardly changed. In the 20 to 30 kDa fraction there was a greater increase in proteins with less electrical charge. The fractioning of the 20 to 30 kDa fraction by cationic exchange of the five grape varieties was analyzed for two years and characteristic profiles were observed for each variety which enables them to be classified according to the fractions obtained. This suggests that it may be a possible tool for characterizing varieties.

This diploma thesis deals with acidic proteins contained in the potato (Solanum tuberosum) tubers, or rather about their separation with chromatography techniques. For the analysis were chosen tubers of two czech potato varieties, Adéla and Westamyl. From the concentrated potato juice were eliminated basic and most of the patatin proteins with the gravity column chromatography. In the next step were applied the chromatography techniques on the anion and hydroxyapatite columns by the fast protein liquid chromatography (FPLC) system. On the anion column were separated protein fractions which contained proteins of molecular weight in the range of 15 kDa to 60 kDa (Adéla) and 15 kDa to 100 kDa (Westamyl). The most abundant were the proteins with molecular weight 15 kDa a 20 kDa. In the last step was used the mass spectrometry for the identification of chosen protein fractions.

Human α-N-acetylglucosaminidase (Naglu) is a lysosomal enzyme implicated in the rare metabolic storage disorder Mucopolysaccharidosis III type B (MPS IIIB). A deficiency in Naglu results in a buildup of heparan sulfate in lysosomes, which is most detrimental in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is currently ineffective in treating the neurological symptoms of MPS IIIB due to the inability of Naglu to cross the blood-brain barrier. This laboratory uses a Spodoptera frugiperda insect cell system to express recombinant Naglu conjugated to a synthetic protein transduction domain with the intent to allow Naglu to cross the blood-brain barrier and treat the neurological symptoms. In the present study, we aimed to purify a recombinant Naglu-PTD4 fusion protein in order to assess its capacity to cross cellular membranes. A three-step method involving multi-modal, hydrophobic interaction, and gel filtration chromatography was optimized to achieve pure Naglu-PTD4, in good yield. Cellular uptake by human MPSIIIB fibroblasts of Naglu-PTD4 was not detectable. It is hypothesized that additional amino acids, including a hexahistidine domain, following the PTD4 domain limited the fusion protein’s membrane transduction capacity. Future studies will focus on removing the additional amino acids and adjusting the purification method as necessary. The ultimate goal of this research is to develop a large-scale recombinant Naglu production protocol for enzyme replacement therapy of MPS IIIB. / Graduate

La nostra proposta ha profunditzat per primera vegada en l’estudi de les lipoproteïnes d’alta densitat (HDL) en la infecció pel virus de la immunodeficiència humana (VIH), i hem determinat la seva implicació en el curs de la infecció i en el desenvolupament de l’arteriosclerosi amb la finalitat de dissenyar noves estratègies terapèutiques. Els nostres resultats mostren com l’estat inflamatori i oxidant persistent associat a la infecció té un paper central en l’aparició d’alteracions metabòliques i el desenvolupament d’aterosclerosi en aquests pacients. Concretament, utilitzant tècniques cromatogràfiques i proteòmiques, mostrem com la presència anòmala de gammaglobulines, molècules pro-inflamatories i reactants de fase aguda en el plasma d’aquests pacients indueixen la modificació del metabolisme, composició i distribució de les partícules HDL, suggerint que la monitorització de les partícules HDL en aquests pacients no ha d’estar basada únicament en la determinació de la concentració del contingut de colesterol, sinó que ha d’incloure la determinació d’altres paràmetres associats a la qualitat, i per tant, a la funcionalitat, de les partícules HDL. / Our proposal has explored for the first time the effect of human immunodeficiency virus (HIV)- infection on high-density lipoprotein (HDL) particles, and we determined their involvement in the course of infection itself and the development of atherosclerosis in order to design novel therapeutic strategies. Our results show how the chronic oxidant and inflammatory status associated with infection plays a central role in the onset of metabolic disorders and the development of subclinical atherosclerosis in these patients. Concretely, using both, chromatographic and proteomic techniques, we observed how the increased levels of gammaglobulin, pro-inflammatory molecules and acute phase reactants in the plasma of these patients induced metabolism alterations and modifications on the distribution and composition of HDL particles, suggesting that the monitoring of HDL particles in these patients should not be only based on the determination of the concentration of cholesterol content, but must include the determination of other parameters associated with particle quality, and therefore the functionality, of HDL particles

Techn. Univ., Diss., 2006--Darmstadt

The diploma thesis is focused on enzyme hydrolysis of potato protein concentrates and fractionation of obtained peptide fragments. Were used protein concentrate from tubers variety Ornella and protein concentrate obtained by swedish company Lyckeby Starch AB. The enzyme hydrolysis lasted 24 hours and were used the proteolytic enzyme alkalasa and trypsin. In this work were prove possitive effect of enzyme hydrolysis on solubility and antioxidative properties of potato protein isolates. The fractionation of obtained peptide hydrolysated was based on systém FPLC (Fast protein liquid chromatography). The fractions contained of peptide fragments about 1, 350 kDa or fragments of smaller moleculary weight. The antixodative activity of subfractions were determIne by method called DPPH. The highest values (2,2 and 2,6 TEAC g/kg) were accured at the subfractions which were separations from Ornella hydrolyzates digeste by enzyme alkalasa.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.

Syftet med arbetet var att försöka utveckla en metod för att isolera lipoprotein(a), ett lipoprotein som korrelerar i hög grad till kardiovaskulär sjukdom, från plasma. Det har varit problematiskt att på ett enkelt sätt separera lipoprotein(a) från LDL på grund av dess strukturella likheter. Som inspiration användes en artikel där separationen utfördes med anjonbyteskromatografi-HPLC (high performance liquid chromatography). Målet var att applicera metoden på ett FPLC-system (fast protein liquid chromatography) med en gelfiltreringskolonn och en anjonbyteskolonn där det kaotropiska saltet natriumperklorat användes som elueringsmedel under anjonbyteskromatografin. Resultaten kontrollerades med DLS (dynamic light scattering), SDS-PAGE och Western blot med antikroppar riktade mot lipoprotein(a). Resultaten visade på goda möjligheter till en bra separation vid fortsatta studier.

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.