[PDF] Protein Interactions And Phase Behavior In Aqueous Solutions eBook

Author : Yu-Chia Cheng
Publisher : ProQuest
Page : pages
File Size : 24,28 MB
Release : 2008
Category : Precipitation (Chemistry)
ISBN : 9780549388180

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Separating proteins in aqueous solutions through fractional precipitation by addition of salts is one of the oldest protein separation methods, and it remains the only purification step for some industrial enzymes and an initial purification step for higher-value therapeutic proteins. However, despite its widespread application, the fundamental characteristics of protein precipitation by salt are still incompletely understood. The primary focus of this thesis is the experimental exploration of the phase behavior of single and binary protein systems during protein precipitation in concentrated ammonium sulfate and sodium chloride solutions, and the effect of solution conditions on protein interactions. Protein precipitation phase behavior of single protein systems was measured as a function of solution conditions for lysozyme and ovalbumin. X-ray powder diffraction (XRD) and optical microscopy were used to characterize the structure of the protein precipitates. These studies showed that crystallization after initial precipitation is feasible even at high ionic strengths if sufficient mixing time is allowed, and such behavior may be a more general phenomenon although different crystallization pathways may be found in some systems and under different conditions; for instance, different pathways are seen in ovalbumin-ammonium sulfate systems as a function of pH. A more important development is the interpretation that emerges of precipitation as a manifestation of a metastable liquid-liquid separation, with the precipitate being a kinetically trapped dense liquid phase. Furthermore, the diversity of possible dense phases (amorphous precipitate, crystal phases or gel) associated with liquid-liquid phase separation shows that understanding the effects of protein interactions of different kinds and at different ranges could have important consequences for the systematic control of protein phase behavior. The phase behavior in selective precipitation of lysozyme-ovalbumin binary mixtures was investigated at various conditions. The best combination of selectivity and recovery of lysozyme was achieved in pH 7 NaCl solutions. At an initial concentration of 30 mg/g H 2 O of lysozyme and 30 mg/g H 2 O of ovalbumin, more than 80% of the lysozyme was collected in precipitate form with 93% purity at ionic strengths above 3 m, while ovalbumin was collected in the supernatant at 80% purity. The purities of both proteins were therefore improved by at least 30% in one step. Moreover, crystallization following initial precipitation was observed in some binary precipitation experiments and indicates that binary precipitation, like single protein precipitation, is a metastable equilibrium state. The observations of selective precipitation behavior were correlated with values of the osmotic second virial coefficient, measured by cross-interaction chromatography (CIC). The results show that interaction measures such as protein self- and cross-interactions can be used as a predictor for general trends and/or inconsistencies in the separation behavior and for optimizing working conditions. Such a mechanistic relationship can obviate the need for extensive trial-and-error methods in order to optimize conditions for protein separations.

Author : Leigh Quang
Publisher :
Page : pages
File Size : 49,15 MB
Release : 2012
Category : Protein-protein interactions
ISBN :

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Protein phase behavior encompasses the formation of dense phases, which include amorphous aggregates, gels, dense liquids, and crystals. The major solution variables that dictate the type of dense phase that is formed are pH, temperature, type of precipitant, precipitant concentration, and protein concentration. Because of the large parameter space and rich variety of phase transitions possible, protein phase behavior is a complex phenomenon. Fundamentally, macroscopic phase transitions are governed by the molecular interactions between proteins in solution. One promising way of quantifying protein-protein interactions and relating them to phase behavior is through the osmotic second virial coefficient B22, a dilute-solution property that characterizes two-particle interactions. The relationship of B22 to overall phase behavior of proteins is explored in this work. The goal of this thesis is to quantitatively relate protein-protein interactions to protein phase diagrams in order to develop predictive models of phase behavior under different solution conditions. A continuum-level approach is used initially to relate experimental B22 data and phase diagrams of proteins by appealing to existing thermodynamic models, with the expectation that a simple continuum model could provide a useful mechanistic framework for predicting protein phase behavior. The first approach attempted was to relate protein interactions and phase behavior within the Flory-Huggins theory of polymer solutions. The second approach utilized the model of Haas and Drenth, which is based on the free energy of mixing for hard spheres. Finally, phase equilibrium was predicted from virial coefficients using the osmotic virial equation. A qualitative relationship was found between B22 and phase behavior from these continuum models; however, quantitative agreement could not be obtained. The isotropic assumption shared among these models in addition to the orientationally-averaged nature of B22 suggests that the anisotropic character of protein interactions cannot be neglected, demonstrating the need for more detailed molecular-level models. The role of anisotropy in protein interactions was explored through analysis of "patch-antipatch" pairs in the computation of B22 in atomistic detail. Patch-antipatch pairs represent highly attractive orientations resulting from geometric complementarity between protein surfaces. Previous work used simple Monte Carlo integration for the calculation of B22 from atomistic models of proteins. However, the presence of patch-antipatch pairs led to significant numerical concerns. These concerns warranted a reexamination of the numerical methods for computing B22. A hybrid Monte Carlo/patch integration approach is utilized to calculate B22 for lysozyme and chymosin B. This method involves a combination of numerical integration techniques in an attempt to obtain better convergence in predicting B22. The overall B22 for the proteins studied was separated into three components: contributions from the excluded volume, from the patch-antipatch pairs, and from background configurations. The excluded volume component was found to be adequately determined using simple Monte Carlo integration. The contributions from individual patch-antipatch pairs were accounted for by carefully integrating the subregions of the configuration space occupied by these pairs using a globally adaptive integration routine. The background component to B22 was also calculated by simple Monte Carlo integration in which the regions of the configuration space occupied by the patch-antipatch pairs were excluded. The calculations performed that account for the full protein structure emphasize the importance of several features of protein interactions. First, the difference in the interaction behavior of the two proteins studied was found to be largely attributed to the charge anisotropy of patch-antipatch pairs. However, the relation of the results to experimental data is limited by the omission of accounting for the specific hydration of proteins. Hydration effects are known to affect, and usually attenuate, patch-antipatch configurations, and therefore would be expected to significantly impact the accurate prediction of B22. Classical colloidal as well as atomistic models that omit these important features are inadequate in providing a quantitative representation of protein interactions for a wide range of solution conditions.

Author : Roger Gregory
Publisher : CRC Press
Page : 596 pages
File Size : 41,1 MB
Release : 1995-01-04
Category : Science
ISBN : 9780824792398

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This work covers advances in the interactions of proteins with their solvent environment and provides fundamental physical information useful for the application of proteins in biotechnology and industrial processes. It discusses in detail structure, dynamic and thermodynamic aspects of protein hydration, as well as proteins in aqueous and organic solvents as they relate to protein function, stability and folding.

Author : Camila Gambini Pereira
Publisher : Academic Press
Page : 684 pages
File Size : 50,18 MB
Release : 2018-10-17
Category : Technology & Engineering
ISBN : 0128115572

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Thermodynamics of Phase Equilibria in Food Engineering is the definitive book on thermodynamics of equilibrium applied to food engineering. Food is a complex matrix consisting of different groups of compounds divided into macronutrients (lipids, carbohydrates, and proteins), and micronutrients (vitamins, minerals, and phytochemicals). The quality characteristics of food products associated with the sensorial, physical and microbiological attributes are directly related to the thermodynamic properties of specific compounds and complexes that are formed during processing or by the action of diverse interventions, such as the environment, biochemical reactions, and others. In addition, in obtaining bioactive substances using separation processes, the knowledge of phase equilibria of food systems is essential to provide an efficient separation, with a low cost in the process and high selectivity in the recovery of the desired component. This book combines theory and application of phase equilibria data of systems containing food compounds to help food engineers and researchers to solve complex problems found in food processing. It provides support to researchers from academia and industry to better understand the behavior of food materials in the face of processing effects, and to develop ways to improve the quality of the food products. Presents the fundamentals of phase equilibria in the food industry Describes both classic and advanced models, including cubic equations of state and activity coefficient Encompasses distillation, solid-liquid extraction, liquid-liquid extraction, adsorption, crystallization and supercritical fluid extraction Explores equilibrium in advanced systems, including colloidal, electrolyte and protein systems