RESUMEN
The aim of this study was to investigate the effect of the addition of different carbohydrates on the thermally induced aggregation of a model globular protein, ß-lactoglobulin (BLG), in the glass state. Amorphous mixtures of BLG with trehalose, sucrose and dextran were prepared by freeze-drying, their glass behaviour was characterised using calorimetry and thermally induced aggregation was measured using size exclusion chromatography. Pure BLG shows increasing levels of aggregation when heated in the temperature range 70-100°C for 48-144 h. The addition of the disaccharides sucrose and trehalose both resulted in a decrease in aggregation rate which approached negligible rates at 50 wt.% carbohydrate. The effect of dextran addition was similar to that of the disaccharides when preparations containing 9wt.% carbohydrate were heated at 70°C for 2 days. However, when the concentration exceeded 23 wt.%, the reaction temperature was 70°C or above or the reaction time was longer than 48 h, the addition of the polysaccharide did not protect the protein from thermally induced aggregation, suggesting that protein-polymer phase separation could have occurred during freeze-drying. Overall the results support the proposal that one aspect of carbohydrate additive functionality is as a diluent with the added condition that the carbohydrate remains miscible with the protein during processing.
Asunto(s)
Dextranos/química , Lactoglobulinas/química , Sacarosa/química , Trehalosa/química , Calorimetría , Cromatografía en GelRESUMEN
It has been reported that caroubin, a protein mixture obtained from carob seeds, has rheological properties similar to those of gluten. Comparative studies of the effects of hydration and temperature on caroubin and gluten were carried out with the aid of NMR, FTIR, scanning electron microscopy, and differential scanning calorimetry techniques. The results show that caroubin has a more ordered structure than gluten and that hydration has little effect on its secondary structure when compared to gluten. Caroubin is more easily accessible to water than gluten, suggesting that caroubin is more hydrophilic in nature. On hydration, caroubin, like gluten, forms fibrillar structures and sheets.
Asunto(s)
Glútenes/química , Polisacáridos/química , Semillas/química , Rastreo Diferencial de Calorimetría , Galactanos , Glútenes/ultraestructura , Espectroscopía de Resonancia Magnética , Mananos , Microscopía Electrónica de Rastreo , Gomas de Plantas , Polisacáridos/ultraestructura , Semillas/ultraestructura , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , AguaRESUMEN
The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.
Asunto(s)
Proteínas de Soja/química , Temperatura , Calor , Microscopía de Fuerza Atómica , Concentración Osmolar , Tamaño de la Partícula , Polímeros/química , Estructura Secundaria de Proteína , Espectrofotometría UltravioletaRESUMEN
The dielectric relaxation behaviour of several amorphous low molecular weight carbohydrates and their 10% w/w water mixtures has been studied in the supercooled liquid and glassy regions in the frequency range 100 Hz to 100 kHz. The dry carbohydrates show a primary alpha-relaxation (activation energy 250-405 kJ mol(-1)) at temperatures above the calorimetric glass transition temperature, Tg, and, in most cases, a secondary beta-relaxation (activation energy 42-55 kJ mol(-1)) at sub-Tg temperatures. Whilst D-mannose showed a beta-relaxation similar in strength to D-glucose, its deoxy sugar, L-rhamnose showed a relatively weak beta-relaxation. This indicates that the hydroxymethyl group influences relaxation in carbohydrate glasses. Addition of water shifted the alpha-relaxations to lower temperatures and increased the strength of the beta-relaxations. In glucitol this resulted in a merging of the alpha- and beta-relaxations. The beta-relaxation increased in strength and decreased in temperature for the series of water mixtures: D-glucose, maltose, and maltotriose.
Asunto(s)
Carbohidratos/química , Agua/química , Rastreo Diferencial de Calorimetría , Estereoisomerismo , Temperatura , TermodinámicaRESUMEN
The glass-transition behaviour of four hydrated wheat gluten proteins (alpha-gliadin, gamma-gliadin, omega-gliadin and high-molecular-weight (HMW) subunits of glutenin) was studied using differential scanning calorimetry (DSC). By fitting the data to the Gordon-Taylor equation, which has previously been used to describe the plasticization of polymers by diluents, the glass-transition temperatures (Tg) for the dry proteins were found by extrapolation. The values for Tg were within the range 397-418 K. Values for the heat capacity increment delta Cp at Tg for the plasticized proteins were also determined and ranged from 0.29-0.47 J g-1 K-1 with no dependence on water content. The differences in glass-transition behaviour of the proteins are discussed in relation to their secondary structure.
Asunto(s)
Gliadina/química , Glútenes/análogos & derivados , Triticum/química , Rastreo Diferencial de Calorimetría , Fenómenos Químicos , Química Física , Cromatografía , Glútenes/química , Estructura Secundaria de Proteína , Temperatura , Termodinámica , Agua/químicaRESUMEN
We have examined the stabilities of the catalytic and binding domains of glucoamylase 1 from Aspergillus niger and how these stabilities are affected by the O-glycosylated linker glycopeptide which separates the domains. On heating, the catalytic domain unfolds irreversibly, whereas the binding domain unfolds reversibly as shown by differential scanning calorimetry and by 1H NMR. The stability of three functional peptides, derived from glucoamylase 1, containing the binding domain alone and with 10 or 38 residues of the linker glycopeptide [Williamson, G., Belshaw, N.J. and Williamson, M. (1992) Biochem. J. 282, 423-428] was examined. Refolding in each case was reversible after thermal or chemical denaturation. beta-Cyclodextrin stabilised the binding domain by the same amount when it was part of glucoamylase 1 or an isolated domain. The thermal stability of the catalytic domain was not affected by the binding domain; however, the catalytic domain increased the melting temperature of the binding domain. Furthermore, the linker glycopeptide stabilised the binding domain against reversible thermal and chemical denaturation by about 10 kJ/mol, but only a portion of the O-glycosylated residues were required for stabilisation. On a simple molecular mass basis, the linker glycopeptide does not contribute as much as expected to the denaturational enthalpy of glucoamylase 1 and, in addition, shows only a small conformational change on chemical or thermal denaturation; this supports an extended structure for the linker. The results demonstrate that the unfolding pathway of glucoamylase 1 depends on the concentration of beta-cyclodextrin and that the presence of the catalytic domain and/or the linker glycopeptide stabilises the binding domain.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Glicoproteínas/química , Desnaturalización Proteica , beta-Ciclodextrinas , Aspergillus niger/enzimología , Rastreo Diferencial de Calorimetría , Ciclodextrinas/farmacología , Glicosilación , Guanidinas/química , Calor , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Relación Estructura-Actividad , TermodinámicaRESUMEN
The melting behaviour of highly crystalline spherulites, of a short chain amylose DP-15 corresponding to both the A and B polymorphs of starch has been studied as a function of water content. At water contents greater than 40% w/w A-type spherulites melt at temperatures approximately 20 degrees C higher than B spherulites, whilst both crystal forms melt at lower temperatures in the presence of increasing amounts of water. Using the Flory-Huggins relationship for polymer crystal melting in the presence of a diluent, it is possible to predict a value for the ideal melting temperature of the dry crystals of 530 K.