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This thesis focuses upon pacidamycin, a member of the uridyl peptide antibiotics, a family of antibiotics which exhibit an, as yet, clinically unexploited mode of action, against MraY. The Goss group has previously demonstrated the ease of accessing N and C-termini analogues of pacidamycin utilizing precursor directed biosynthesis. The central diamino acid is key to pacidamycin's activity, yet little work has been carried out, to date, to investigate the SAR around this moiety. Particularly this thesis describes work toward generating pacidamycin analogues using the complementary tools of organic synthesis and biosynthesis. Chapter 1 introduces natural compounds and their importance in clinical use, provides a brief overview of the history of antibiotics and focuses on the urgent need for new antibiotics displaying new chemical architectures and possessing novel modes of action. This chapter also introduces uridyl peptide antibiotics and overviews the SAR studies around these unusual peptides, focusing on pacidamycin in particular. Diaminobutyric acid is central to these structures and a discussion of a selection of published methods to synthesis α, β-diaminobutyric acid (DABA) is also presented. Chapter 2 describes the synthesis of DABA and two analogues, in which the C-methyl moiety has been substituted by an ethyl or a cyclopropyl group. The mutasynthesis approach utilised in the attempt to generate novel pacidamycins and discussion around the results observes is also described. Chapter 3 demonstrates a three step one-pot reaction to access 1,3-disubstituted urea molecules. The chapter starts with a brief overview of previously established methods in the literature to access these useful molecules, and then moves towards a discussion about the reaction optimisation. The chapter also describes a family of analogues generated utilising this novel approach; and exploring the use of these analogues in the mutasynthesis of pacidamycin. In order to access the desired pacidamycin analogues with the modified diamino acid residue, it was determined that it is currently not possible to use a mutasynthesis approach, instead an approach of total synthesis needed to be employed. Chapter 4 describes this total synthesis. The C- terminal urea motif was generated using a novel 1-pot phosphine free route developed during this study. To access the central native (2S, 3S)- DABA, a variation of the route of Merino et al's via Garner's aldehyde was initially utilised. Subsequently, a shorter and more flexible approach from Soloshonok et al via a Ni (II) Schiff base complex of glycine was adopted. Unpublished results from the Goss group have shown that the 2',3'dihydroxy uridine analogues in pacidamycin conferred broader spectra of activity. Work towards the synthesis of these analogues has been conducted. The order of assembly of the peptide and the nucleoside fragments was in alignment with Boojamra et al's approach. If the de-protection chemistry had worked according to plan, this would have resulted with a synthesis that is at least 6 steps shorter and higher yielding then Boojamra's. The introduction in this chapter reports the various methods previously reported in the literature for the total synthesis of pacidamycin. A discussion about the current progress in the total synthesis highlighting the difficulties faced is also shown. Chapter 5 demonstrates utilising semi-synthesis as a useful tool to generate novel pacidamycins by applying a Pictet-Spengler reaction on pacidamycin 4. This chapter starts with an overview of this phosphate mediated Pictet-Spengler reaction. In addition, a discussion about the large-scale fermentation of Streptomyces coeruleorubidus, the wild type producer of pacidamycin, and the generation of pacidamycin analogues utilising a semi-synthesis approach is also presented. Chapter 6 describes the future work following on from this study building upon each of the above chapters.

Stringent pollutant regulations on diesel-powered vehicles have resulted in the development of new technologies to reduce emission of nitrogen oxides (NOx). The urea Selective Catalyst Reduction (SCR) system and Lean NOx Trap (LNT) have become the two promising solutions to this problem. Whilst the LNT results in a fuel penalty due to periodic regeneration, the SCR system with aqueous urea solution or ammonia gas reductants could provide a better solution with higher NOx reduction efficiency. This thesis describes an experimental investigation which has been designed for comparing the effect NOx abatement of a SCR system with AdBlue urea spray and ammonia gas at 5% and 4% concentration. For this study, a SCR exhaust system comprising of a diesel particulate filter (DPF), a diesel oxidation catalyst (DOC) and SCR catalysts was tested on a steady state, direct injection 1998 cc diesel engine. It featured an expansion can, nozzle and diffuser arrangement for a controlled flow profile for CFD model validation. Four different lengths of SCR catalyst were tested for a space velocity study. Chemiluminescence (CLD) based ammonia analysers have been used to provide high resolution NO, NO2 and NH3 measurements across the SCR exhaust system. By measuring at the exit of the SCR bricks, the NO and NO2 profiles within the bricks were found. Comparison of the measurements between spray and gas lead to insights of the behaviour of the droplets upstream and within the SCR bricks. From the analysis, it was deduced that around half to three quarters of the droplets from the urea spray remain unconverted at the entry of the first SCR brick. Approximately 200 ppm of potential ammonia was released from the urea spray in the first SCR brick to react with NOx. The analysis also shows between 10 to 100 ppm of potential ammonia survived through the first brick in droplet form for cases from NOx-matched spray input to excess spray. Measurements show NOx reduction was complete after the second SCR bricks. Experimental and CFD prediction showed breakthrough of all species for the short brick with gas injection due to the high space velocity. The long brick gas cases predictions gave reasonable agreement with experimental results. NO2 conversion efficiency was found higher than NO which contradicts with the fast SCR reaction kinetics. Transient response was observed in both cases during the NOx reduction, ammonia absorption and desorption process. From the transient analysis an estimate of the ammonia storage capacity of the bricks was derived. The amount of ammonia slippage was obtained through numerical integration of the ammonia slippage curve using an excel spreadsheet. Comparing the time constant for the spray and gas cases, showed a slightly faster time response from the gas for both NOx reduction and ammonia slippage.

Thesis (MScAgric)--Stellenbosch University, 1999. / ENGLISH ABSTRACT: The rate of hydrolysis of urea in the rumen of animals is a major limitation when considering the substitution of natural protein with urea in the formulation of rations. The aim of this study was to evaluate polymer coated urea prills with variable coating thickness and evaluate its potential as a slow-release NPN compound. A new slowrelease urea compound, made by coating prilled feedgrade urea with a co-polymer of urea-formaldehyde resin and a castor-coconut alkyd was initially evaluated for urea-nitrogen concentration in distilled water in order to evaluate its potential as a slow-release urea product for ruminants. Amino/alkyd or polyester blends are among the cheapest of the modern synthetic systems and are considered because it is non-toxic, low-cost, biodegradable and easy to manufacture. A 2 x 2 x 2 x 2 factorial design was used and 16 individual products were made and evaluated. The Wurster method was used to encapsulate urea prills. The slopes of the urea release curves represented the release rate of the encapsulated products and were compared to identify the process variables, which had an effect on release rate. Two of the coating variables, coating weight and alkyd: resin ratio, had a major effect (P = 0.0001) on the release rate of urea. The crushing strength of encapsulated products was significantly (P = 0.0001) higher than that of untreated urea. Results motivated the evaluation of the products in the rumen of sheep in terms of rumen ammonia and blood urea N concentrations. Four slow-release products were made after interpreting results from the first study, and differed on account of the coating weight and the composition of the co-polymer. Fifteen fistulated wethers were randomly allotted into 5 groups and intraruminally received an equivalent of 15g urea. Rumen ammonia and blood ammonia were taken at 0, 1, 2, 4, 6, 8, 12, 16, 20, 24, 36 and 48 h after administration of the various treatments. Slow release urea (SRU) resulted in significantly lower rumen ammonia peaks (P = 0.0001) than untreated urea, while the peaks were also significantly delayed. Untreated urea resulted in the maximum concentration at two hours after administration of the urea (P = 0.0685) while the SRU's reached a maximum at six hours after administration in the rumen. No significant differences between the four different SRU types were found. Responses in blood urea-N was similar to that observed for rumen ammonia nitrogen. The encapsulation was effective in decreasing the rate of ammonia release from the urea for up to six hours after administration. In a third trial four Dehne Merino wethers were used in a 2 x 2 Latin square design. They received a SRU product equivalent to 0.4 g urea per kg body weight orally. Rumen liquor and blood samples were taken at 0, 1, 2, 4, 6, 8, 12, 16, 20, 24, 36 and 48 h after intake. Difficulty was initially experienced with ingestion and palatability of the SRU products due to the strong formaldehyde and butanone odour present in the coating. The maximum rumen ammonia (NH3) concentration for the SRU were lower than that of untreated urea (17.5 mg N/dl vs. 66.9 mg N/dl). The time to reach blood urea levels also differed considerably (6 h vs. 24 h for blood urea nitrogen) between treatments. The encapsulation of urea prills shows potential solutions to reduce the solubility of urea and also reduce the hygroscopic nature of urea and improve the palatability and storage characteristics thereof. Keywords: Slow-release urea, encapsulate, copolymer, urea formaldehyde rumen ammonia, blood urea nitrogen, solubility, palatability, storage characteristics. / AFRIKAANSE OPSOMMING: Die vinnige tempo waarteen ureum in die rumen na ammoniak omgesit word, is die grootste beperking in die optimale benutting van ureum, as vervanging van natuurlike protein in herkouerrantsoene. Die doel van hierdie studie was om 'n stadig vrystellende ureumproduk te ontwikkel wat die rumenammoniakvlak gedurende 'n aansienlike periode van die dag bokant 'n sekere vlak kan hou. 'n Nuwe stadig vrystellende ureumproduk, vervaardig deur ureumkorrels met 'n kopolimeer van ureum-formaldehiedhars en 'n kastor-en klapperalkied te bedek, is geevalueer om die potentiaal as stadig vrystellende nie-protein stikstof (NPN)-produk vir herkouers te ondersoek. Die veiligheid, biodegradeerbaarheid, lae koste en maklike vervaardiging van amino/alkied-kopolimere maak dit een van die goedkoopste sintetiese sisteme om vir stadig vrystellende sisteme te oorweeg. 'n 2 x 2 x 2 x 2 Faktoriale antwerp is gebruik om 16 individuele produkte te vervaardig. Die Wurster-metode is gebruik om individuele korrels te enkapsuleer met die polimeer en die potentiaal van die produkte is aanvanklik geevalueer deur die ureumstikstofvrystelling in gedistilleerde water te meet. Die hellings van die vrystellingsgrafieke is vergelyk om die veranderlikes te bepaal wat die grootste invloed op die vrystellingstempo van ureum uit die ge·inkapsuleerde produkte het. Resultate dui dat twee verandelikes 'n betekenisvolle effek het op die vrystellingstempo, nl. dikte van die omhulsel, en die samestelling van die kopolimeer (P = 0.0001 en P = 0.0135, onderskeidelik) het. Die samedrukbaarheid van die ge ·lnkapsuleerde produkte was ook betekenisvol hoer (P = 0.0001) as die van onbehandelde ureumkorrels, wat lei tot verbeterde bergings- en hanteringseienskappe. lnterpetering van resultate lei tot die vorming van vier stadig vrystellende produkte. Vyf groepe van 3 volwasse rumengefistuleerde Dohnemerinohamels is in 'n proef gebruik om die potentiaal van die produkte verder te ondersoek. 'n Ekwivalent van 15 g ureum is direk in die rumen van elke dier geplaas en ammoniak-en bloed monsters is 0, 1, 2, 4, 6, 8, 12, 16, 20, 24, 36 & 48 ure na dosering geneem. Die stadig vrystellende ureumprodukte het 'n betekenisvol laer maksimum waardes vir beide ammoniak-en bloedstikstofureum getoon (P = 0.0001 ). Onbehandelde ureum het 'n maksimum rumenammoniakstikstof konsentrasie reeds twee ure na toediening bereik in vergelyking met ses ure vir die stadig vrysellende produkte. Geen betekenisvolle verskille in hierdie parameters is tussen die ge·lnkapsuleerde produkte gevind nie, terwyl geen interaksie is tussen hoofeffekte voorgekom het nie. In 'n derde proef is vier Dohnemerinohamels gebruik om die vrystellingstempos, in terme van rumenammoniak-en bloedureumstikstof te bepaal waar die stadig vrystellende produk en onbehandelde ureum direk aan die diere gevoer is . 'n Ekwivalent van 0.4 g ureum/kg liggaamsmassa is gevoer. Aanvanklik is inname- en smaakliksheidprobleme ondervind, moontlik as gevolg van die sterk butanoon-en formaldehiedreuk van die omhulsel. Die rumenammoniakstikstof het 'n laer maksimum (17.5 vs. 66.9 mg N/1 00 ml) as die van onbehandelde ureum gehad terwyl die tyd wanneer maksimum konsentrasie bereik word ook aansienlik later was. Die polimeer inkapsulering van ureumkorrels toon potensiaal as 'n stadig vrystellende ureumproduk deurdat dit die oplosbaarheid van ureum in die rumen verlaag. Bykomende voordele is dat die omhulsel die higroskopisiteit verlaag en die samedrukbaarheid verhoog, beide eienskappe wat die hantering-en bergingseienskappe bevorder. Sleutelwoorde: Stadig vrystellende ureum, enkapsulering, kopolimeer, ureumformaldehied, rumenammoniak, bloed ureum stikstof, oplosbaarheid, smaaklikheid, bergingseienskappe.

Nitrogen (N) loss in the form of volatilized ammonia (NH₃) is a considerable problem when ammonium (NH₄⁺) forming fertilizers are applied to calcareous or alkaline soils. Large areas of agricultural land, contain alkalinity and salinity problems, are potentially suitable for crop production with little alteration. This study was conducted to determine and compare the effectiveness of urea phosphate (UP) in reducing soil alkalinity and NH₃ loss. The volatilization of NH₃ from UP and urea (U) was studied on 3 selected soils (Hayhook SL, Laveen L and Latene L) using an aeration system. Urea phosphate and U were each applied at rates of 0, 50, 100 and 200 ppm-N either to the surface dry or in solution or mixed with the soil. The volatilized NH₃ was trapped in sulfuric acid, sampled periodically and analyzed for N using the semi microkjeldahl distillation apparatus. The effect of UP, Sulfur-Foam (SF), Phosphuric Solution (PHP) and a mixture of SF and UP (Mix) on leaching soil sodium (Na) and salinity was also studies on two soils (Pima L and Crot CL) in columns. Each of these amendments was applied at a rate of one and two equivalent amounts of the exchangeable Naₑₓ. The highest N loss in the form of NH₃ occurred when U was applied to Hayhook soil. However, UP applied to Hayhook soil (neutral to acidic, coarse textured and low CaCO₃ content) resulted in the lowest NH₃-N loss. Less NH₃-N loss was found from U application to Laveen and Latene soils (fine textured with higher CaCO₃ content) than with Hayhook soil. The general trend was higher N loss, in the form of volatilized NH₃, with surface application dry or in solution than when mixed with the soil. This trend showed an increase in the amount of volatilized NH₃ with increasing rate of N application. Urea phosphate was as effective as PHP or Mix (acid containing fertilizers) treatments in reducing soil salinity and alkalinity in Pima and Crot soils. No difference was found between rates of application (1 and 2 equivalent amount of Naₑₓ) except for soil pH. A similar trend in the decrease in soil salinity was found to that of the pH which was in the order PHP, UP, Mix, SF and control treatments. No significant difference was found between SF and control treatments in all parameters. No significant difference was found between treatments for exchangeable Ca. This was affected by the Ca compounds present in the soil. Generally, UP is a potential fertilizer for supplying N and phosphorus (P) as plant nutrients, reducing NH₃ volatilization, and can be used as a soil amendment to control soil salinity and alkalinity.

SETD2/HYPB (SET domain-containing protein 2 / Huntingtin yeast partner B) is the predominant lysine methyltransferase in mammals that mediates histone H3 lysine-36 (H3K36) trimethylation, which is associated with transcription elongation and RNA splicing. SETD2 is further implicated in p53 function, vascular development, cancer progression and, through Huntingtin-interaction, Huntington's disease. Although different transcripts and putative protein isoforms have been detected previously, their identity, function and significance have not been rigorously investigated. This thesis aims to identify and characterise endogenous transcripts and protein isoforms of SETD2 in mouse fibroblasts. Affnity-purified N- and C-terminal antibodies specifically detected the ≈ 290 kDa methyltransferase (p290^SETD2), verified by RNAi, in addition to N terminal-specific ≈ 120 kDa protein, and C terminal-specific forms at ≈ 140 and ≈ 66 kDa (p66), which all appeared too stable to deplete by transient siRNA transfection. Conserved in human and mouse cells, immunodetection of p66 exhibited unusual requirement for denaturation with urea at 95°C. Subcellular fractionation revealed distinct extraction properties of putative isoforms and facilitated partial purification of p66 for proteomic analysis. Co-fractionation and co migration by two-dimensional gel electrophoresis of p66 detected by two independent C terminal antibodies suggested it represents a novel C terminal-specific isoform. Reverse transcription−PCR and DNA-sequencing demonstrated the existence of multiple, alternatively-spliced Setd2 transcripts that plausibly generate truncated proteins. A transcript variant containing a novel complete open-reading-frame, consistent for p66 generation, was identified. Its ectopic expression in mouse fibroblasts produced a distinct SETD2 isoform, whose physical and extraction characteristics were studied in comparison with endogenous immunoforms. In summary, this thesis demonstrates that multiple alternatively-spliced transcripts arise from the Setd2 gene, consistent with immunodetection of several C- and N-terminal-specific putative SETD2 isoforms, additional to the H3K36 methyltransferase. Verification of these isoforms by independent methods would have implications for proposed interactions and function of SETD2 in transcription, epigenetics, cancer development and Huntington’s disease.

The objective of this Ph.D. study was to develop new and improved miniaturized particle-based optochemical sensors for the analysis of biological fluid and cellular components. This is highly important because current sensing systems can be biologically toxic and incompatible, invasive, and have limited responsiveness. To accomplish this goal we defined three tasks. The first was to develop lipobead-based sensors for chloride. The halide-specific fluorescence dye, lucigenin, was immobilized into the phospholipid membrane of the lipobeads to enable chloride ion detection. The fluorescence intensity of lucigenin decreases with increasing chloride ion concentration due to dynamic quenching. To stabilize the lipobeads we co-immobilized hexadecanesulfonate molecules into the phospholipid membrane. We also immobilized the chloride ionophore [9] mercuracarborand-3 (MC-3) into the lipobeads membrane. The study resulted in a unique submicrometric chloride ion sensor, which is suitable for chloride ion measurements in biological fluids. The second task was to develop for the first time lipobeadbased biosensors. Urea was chosen as a model substance since the urea/urease biosensing system is well known. Fluorescence sensing lipobeads were characterized by coating carboxylfunctionalized silica microspheres with phospholipids for the measurement of urea in aqueous samples. The enzyme urease and the pH indicator Fluorescein-5-thiosemicarbazide were attached covalently to the phospholipid membrane of the lipobeads. We prepared improved fluorescence sensing lipobeads by utilizing covalent chemistry to bind the phospholipid membrane to the silica particles and the fluorophores to the membrane. It led to improvement in the stability of the newly developed urea sensing lipobeads compared to previously developed micrometric fluorescence sensors. The final task of this study was to coat particle-based sensors with cell penetrating peptides to enable their permeation into cells. This step is essential for the use of particles as intracellular sensors. Streptavidin coated microspheres were modified by the strongest noncovalent interaction between avidin and biotin. Tat peptide and nonfluorescence indicator flubida were attached to the surface of the microspheres. These nanoparticles were delivered into MCF7 and Hela cancer cells for pH measurement. Before penetrating into the cells, flubida did fluoresce in cell medium; however it did not convert to fluorecein in Phosphate Buffered Saline (PBS) buffer.

The solvent-free (Cp*IrCl2)2 catalyzed N-alkylation of urea with benzyl alcohol has been studied. A variety of reaction conditions were studied and optimized to produce a high yield (82%) of N,N-dibenzylurea. A series of substituted benzyl alcohols were examined at the optimal reaction conditions. However, the preparation of substituted benzyl urea derivatives using conditions optimized for benzyl alcohol gave poor yields or intractable mixtures.

A hemoglobina de Glossoscolex paulistus (HbGp) é caracterizada por uma massa molecular de 3,6 MDa, alta estabilidade oligomérica, resistência a auto-oxidação, e alta afinidade em ligar oxigênio. A estrutura quaternária desta macromolécula apresenta 144 cadeias com grupo heme (globinas) e 36 cadeias sem grupo heme (linkers), dispostos em duas camadas hexagonais. No presente trabalho estudos de caracterização das subunidades da HbGp, bem como da estabilidade da HbGp em diferentes formas, em função do pH, e em diferentes concentrações de ureia, por diferentes técnicas biofísicas, foram realizados. Os estudos de caracterização por eletroforese SDS-PAGE, MALDI-TOF-MS e ultracentrifugação analítica (AUC) das subunidades isoladas mostraram que apenas o monômero d obtido da cromatografia de exclusão por tamanho (SEC) tem alto grau de pureza. Para as demais frações mais de uma contribuição foi observada em solução. Assim, para a fração trimérica, duas espécies estão presentes em solução, a espécie predominante (87 %) é atribuída ao trímero abc e a outra espécie (13 %) pode ser associada ao complexo (abc + L). Os dados espectroscópicos e de AUC mostraram que a estabilidade da HbGp depende fortemente do estado de oxidação do heme, do ligante coordenado no centro metálico e da concentração de proteína. Assim, a forma oxidada, a meta-HbGp, mostrou-se menos estável em meio alcalino e na presença de ureia, seguida pelas formas oxi- e cianometa-HbGp. Desta forma, no pH 8,0, a meta-HbGp está totalmente dissociada em trímero abc e monômero d, enquanto a oxi-HbGp está apenas parcialmente dissociada com uma contribuição de 88 % de proteína íntegra em solução e a cianometa-HbGp não sofre dissociação oligomérica. Os valores de coeficiente de sedimentação s20,w e massa molecular (MM) determinados para as espécies em solução são similares aos observados para as correspondentes espécies isoladas por SEC. Na presença de ureia a mesma tendência foi observada para as três formas da HbGp. Porém, para uma caracterização melhor de processo de desnaturação, os dados espectroscópicos foram analisados usando modelos de dois e três estados para obter informações sobre os parâmetros termodinâmicos do sistema. Assim, bons ajustes foram obtidos usando ambos os modelos, no entanto, o modelo de três estados foi mais adequado para descrever o processo. Por este modelo o processo de desnaturação da HbGp pode ser descrito por duas etapas. A primeira etapa, na faixa de 1,0 - 3,0 mol/L de ureia, está associada a transição do estado nativo para o estado intermediário (N → I), e é caracterizada pela dissociação do oligômero nas diferentes subunidades da HbGp. O estado intermediário apresenta propriedades físico-químicas similares ao estado nativo, sugerindo que o processo de dissociação oligomérica não induz mudanças significativas na estrutura secundária e na região do grupo heme da proteína. Os parâmetros termodinâmicos associados à primeira transição apresentaram erros consideráveis, que podem ser atribuídos à complexidade do estado intermediário com diferentes espécies em solução bem como à semelhança ao estado nativo. A segunda etapa (I → U) com transição bem definida entre 4,5 - 5,0 mol/L de desnaturante é caracterizada pela desnaturação das subunidades dissociadas. Os dados de AUC e SAXS são consistentes com os dados obtidos por espectroscopia, onde a primeira etapa do processo foi caracterizada pela dissociação oligomérica do oligômero em dodecâmero (abcd)3, tetrâmero abcd, trímero abc e monômero d. Para concentrações acima de 4,0 e 5,0 mol/L de ureia, para oxi-HbGp e cianometa-HbGp, respectivamente, aumentos significativos nos valores de I(0), Dmax e Rg sugerem que as subunidades da HbGp estão desnaturadas em solução. As massas moleculares (MM) obtidas por espectrometria de massas e AUC, e os coeficientes de sedimentação s20,w são consistentes com outros resultados reportados para hemoglobinas ortólogas. Além disso, os resultados aqui apresentados representam um avanço importante na caracterização do processo de desnaturação de proteínas oligoméricas complexas. / Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3.6 MDa, a high oligomeric stability, a high resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this current work the characterization of the HbGp subunits and the effect of pH and urea upon the oligomeric stability were studied by several biophysical techniques. Our results obtained by electrophoresis SDS-PAGE, MALDI-TOF-MS and analytical ultracentrifugation (AUC) showed that only the monomer d isolated by size exclusion chromatography (SEC) presented high purity. For the other fractions various species were observed in the solution. Thus, for the trimeric fraction, two species are present in the equilibrium, the main species with percentage contribution of 87 % is assigned to the trimer abc and the species with 13 % in the solution is associated to the complex (abc + L). Additionally, the data obtained by several spectroscopic techniques and AUC show clearly that the oligomeric stability of HbGp depends on the iron oxidation state, the specific ligand coordinated to the iron and the protein concentration. Therefore, our results show that the met-HbGp form is the less stable one in the alkaline medium and in the presence of urea, followed by the oxy- and cyanomet- forms. In this way, at pH 8.0, the met- form is fully dissociated into smaller subunits, such as, trimer abc and monomer d, while the oxy-HbGp is partially dissociated with a significant percentage contribution (88 %) of undissociated protein, and the cyanomet-HbGp does not undergo oligomeric dissociation. The sedimentation coefficients (s20,w) and molecular masses (MM) values for species present in the solution, at different pH, are very close to the values obtained for isolated species. In the presence of urea the same behavior was observed for the three HbGp forms as compared to the alkaline medium. However, for a full characterization of the unfolding process the thermodynamic parameters were obtained by spectroscopic data analysis using models of two and three states. Adequate fits were obtained for both models, but the three states model was very appropriate to describe the HbGp denaturation process. Thus, the denaturation process of HbGp is defined by two phases. The first phase between 1.0 and 3.0 mol/L, of urea is assigned to the transition of native state to an intermediate state (N → I), and is characterized by dissociation of the oligomer in several subunits. The strong similarity of the intermediate state to the native one suggests that oligomeric dissociation induces little changes in the secondary structure and the region of heme group of the protein. As a consequence, the thermodynamic parameters associated to the first transition have large errors due to the complexity of the intermediate state with different species in the solution, as well as its great similarity to the native state. The second phase (I → U), associated with a cooperative transition at 4,5 - 5,0 mol/L of denaturant agent, is attributed to the unfolding of the dissociated subunits. Our AUC and SAXS data are very consistent with spectroscopic data. Thus, in the first phase the oligomeric dissociation of whole protein in dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d was observed. For urea concentrations above 4.0 - 5,0 mol/L, for oxy-HbGp and cyanomet-HbGp, respectively, the significant increase in I(0), Dmax and Rg values suggests that the HbGp subunits are denatured in the solution. The molecular masses values (MM) obtained by mass spectrometry and AUC, and the sedimentation coefficients (s20,w) are consistent with others results reported in the literature for orthologous hemoglobins. In addition, the results of this work correspond to an important advance in the characterization of the denaturation process of this complex oligomeric protein.