A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate << trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH, ionic strength, and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol/l HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery. / Graduate

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Hydrophilic interaction chromatography is well known as a powerful technique separation of polar and ionizable compound nowadays. However the retention mechanism of the technique is still under debate. Understanding retention mechanism would facilitate the method development using the technique and its future improvement. This was inspiring and became the goal of this thesis. This work involves the characterization of the water enriched layer regarding to water and buffer salt accumulation. Twelve HILIC stationary phase with a diverse surface chemistry regarding to function groups and modification type were studied. Effect of water and salt on regarding to the retention mechanism was investigated by correlating the adsorption data to the retention of selected solutes This also involved the characterization of interactions involve in the separation of 21 HILIC columns. Interactions was probe by retention ratio of pair solutes which are characteristic for each specific interaction. The data was evaluate using principle component analysis – a multivariable data analysis method. The model was comprehensive and its outcomes were confirmed by the studies on adsorptions of water and salts.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.

There is a strong link between solubility, and thus crystallisation, and the molecular interactions of proteins in dilute salt solutions. Such molecular interactions are governed by the weak interaction forces (electrostatic, hydration and hydrophobic). Such forces can be quantitatively estimated in terms of a second virial self-coefficient (B22) and a second virial cross-coefficient (B23) for a single and a binary protein system, respectively. Previous studies confirmed the relation between a value of the second virial coefficient and a type of interaction (attractive or repulsive). The aim of this thesis is to correlate the second virial coefficient with the solubility and nucleation for single and binary protein systems. Model proteins used in this work are lysozyme and ovalbumin from egg-white, and α-amylase from Bacillus Licheniformis (BLA). The measurements are performed for sodium chloride and ammonium sulphate solutions in an acidic pH at 20 oC. Interaction chromatography is used in this work to estimate the B22 and B23 values for the model proteins in salt solutions. From the measured values of B22 and B23, the type of interaction is generalised as a function of the salt type, salt concentration, pH and protein type. For the single protein systems, in ammonium sulphate solutions (0.1 - 2.4 M) at pH 4.0 and 7.0, repulsion or no interactions are observed below 0.8 M and, as the salt concentrations are increased attractive self-interactions are observed for the model proteins. However, for the sodium chloride solutions (0.1 - 2.0 M) at pH 4.0 and 7.0, the interaction patterns vary with the salt concentration, the pH and the type of protein studied. A common feature of the self-interaction for all the model proteins is the attractive interactions close to the isoelectric point. For the binary protein systems, three distinct regions are observed in the ammonium sulphate solutions (0.1 - 1.6 M) at pH in the range 4.0 - 7.0. Attractive or no cross-interactions are observed at low salt concentrations (< 0.5 M). At the intermediate salt concentrations (0.5 - 1.0 M), the cross-interactions are constant and near zero. This is followed by a sharp increase in the attractive interactions above 1.0 M ammonium sulphate concentrations. However, for sodium chloride solutions (0.1 - 1.6 M) at pH 4.0 - 7.0, two distinct regions are observed. Attraction or no interactions are observed at low salt concentrations (< 0.5 M) and above 0.5 M concentrations of sodium chloride, negligible cross-interactions are observed between model proteins. For the single protein system, an overall increase in the solubility of three model proteins is observed with an increase in the concentrations of ammonium sulphate and also for sodium chloride solutions except for BLA, where a salting-in behaviour is observed. Linear regression is used on the solubility data to determine the parameters of the Cohn equation (β and Ks) where the values of β vary with solution pH, protein type and salt type. The values of Ks vary with protein type and salt type. However, it is insensitive to the solution pH for lysozyme in ammonium sulphate, ovalbumin in sodium chloride and BLA in ammonium sulphate solutions. For the binary protein system, the presence of ovalbumin had a measurable effect on lysozyme solubility at pH < 5.0 in both salts. In low concentration sodium chloride solutions (< 0.3 M), a decrease in the solubility of lysozyme was observed with the presence of ovalbumin at acidic pH < 5.0. However, in ammonium sulphate solutions, the lysozyme solubility increases with the addition of ovalbumin in the salt concentration range 1.6 - 2.0 M and at pH < 4.0. The primary nucleation threshold values are also determined for lysozyme in sodium chloride and ammonium sulphate solutions. In sodium chloride solutions (0.2 - 1.0 M), the critical supersaturation values increase as the solution pH is raised from 4.0 to 7.0; however in ammonium sulphate solutions (1.0 - 2.0 M), the reverse effect is observed. The critical supersaturation required to nucleate lysozyme in ammonium sulphate solutions is approximately three times higher than in sodium chloride solutions. For the single protein systems, the measured values of solubility and B22 were correlated using published models (RSL and HDW). For each protein-salt combination, a reasonable single correlation between solubility and B22 is possible as the salt concentrations and pH are varied. There are separate correlations for sodium chloride and ammonium sulphate solutions. Based on the correlation curve of solubility and B22, it is proposed that the acidic pH range (4.0 - 5.0) is better for crystallising and precipitating globular proteins from these salt solutions. If the values of solubility and B22 are converted into a non-dimensional quantity, the data derived from the different protein-salt systems collapse onto a single curve for the same salt type. The B22 values are also correlated with the critical supersaturation (ln(c*/S)) for the primary nucleation of lysozyme in salt solutions. The values of the critical supersaturation increase as the values of the second virial coefficient become negative or reduce. The ideal critical supersaturation required to create nuclei of lysozyme in salt solutions is between 0.1 and 1.4. For the binary protein systems, B23 values were related to the slope of the lysozyme and ovalbumin plot at same salt concentration and solution pH. Further work is required for binary protein systems to generalise such correlations as a function of the salt concentration and pH. The correlations derived in this thesis are useful generally to predict the solubility and primary nucleation of globular protein in salt solutions. This work reinforces the importance of the second virial coefficient in predicting the crystallisation of protein in salt solutions.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.

Ces travaux de thèse concernent les développements d'une méthode de séparation chromatographique couplée simultanément à l'ESI-MS et l'ICP-MS afin de réaliser l'analyse de spéciation exhaustive des lanthanides en phase aqueuse représentative des phases de désextraction des procédés de traitement du combustible usé. Cette méthode analytique permet de séparer, caractériser et quantifier des complexes de lanthanides à ligands polyaminocarboxyliques comme le DTPA et l'EDTA, utilisés comme agents complexants dans ces procédés. La méthode de séparation par chromatographie HILIC des complexes de lanthanides a été mise au point avec la phase stationnaire à fonctions amide. Un criblage d'une large gamme de compositions de phase mobile a permis de déterminer que le mécanisme d'adsorption est prédominant lors l'élution des complexes de lanthanides et d'obtenir des conditions de séparation optimisées. Des conditions d'analyse plus rapides obtenues avec une colonne à fonctions amide de granulométrie sub-2 µm et de longueur plus faible ont permis de réduire le temps d'analyse d'un facteur 2,5 et la consommation de solvant de 25 %. La caractérisation structurale et isotopique par HILIC ESI-MS a été réalisée ainsi que la mise au point d'une méthode d'étalonnage externe. Les performances analytiques de la méthode de quantification ont été déterminées. Enfin, le développement d'un système de couplage de l'HILIC à l'ESI-MS et l'ICP-MS a été réalisé. Une méthode de quantification simultanée par ESI-MS et par ICP-MS a permis de déterminer la distribution quantitative des espèces en solution ainsi que les performances analytiques associées. / This work focuses on the development of a chromatographic separation method coupled to both ESI-MS and ICP-MS in order to achieve the comprehensive speciation analysis of lanthanides in aqueous phase representative of back-extraction phases of advanced spent nuclear fuel treatment processes. This analytical method allowed the separation, the characterization and the quantitation of lanthanides complexes holding polyaminocarboxylic ligands, such as DTPA and ETDA, used as complexing agents in these processes. A HILIC separation method of lanthanides complexes has been developed with an amide bonded stationary phase. A screening of a wide range of mobile phase compositions demonstrated that the adsorption mechanism was predominant. This screening allowed also obtaining optimized separation conditions. Faster analysis conditions with shorter amide column packed with sub 2 µm particles reduced analysis time by 2.5 and 25% solvent consumption. Isotopic and structural characterization by HILIC ESI-MS was performed as well as the development of external calibration quantitation method. Analytical performances of quantitation method were determined. Finally, the development of the HILIC coupling to ESI-MS and ICP-MS was achieved. A simultaneous quantitation method by ESI-MS and ICP-MS was performed to determine the species quantitative distribution in solution. Analytical performances of quantitation method were also determined.

In frame work of this diploma thesis, monolithic stationary phases based on hydroxymethylmethacrylate were prepared in fused silica capillaries of 320 μm innerdiameter. Monolithic columns were synthesized by a simple procedure using a polymerization mixture, consisting of a monomer N-(hydroxymethyl) methacrylamide (HMMAA), a croslinking agent ethylene dimethacrylate (EDMA), porogenic solvents butane- 1,4-diol, propane-1-ol and an initiator α,α′-azobisisobutyronitrile (AIBN). Prepared HMMA monolithic columns were utilized for separation of mixtures of biologically active compounds, namely peptides with small number of amino acids. Mechanical strength and specific permeability were determined for selected monolithic columns. Keywords: HPLC, HILIC mechanism, hydroxymethyl methacrylate (HMMA) monolithic columns, amino acid, enkephalins.