Affecting Protein Solubility

As in any crystallization, the production of protein crystals requires bringing the protein into a supersaturated liquid state. The degree of supersaturation determines the rate of nucleation as well as crystal growth rate. Each of these phenomena are 

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The above example also gives an indication of the relative importance of carbohydrate analysis. Without question, protein glycosylation is the most complex of all posttranslational modifications made in eukaryotic cells, the importance of which cannot be underestimated. For many compounds, glycosylation can readily affect protein solubility (as influenced by folding), protease resistance, immunogenicity, and pharmacokinetic/pharmacodynamic profiles (i.e., clearance and efficacy) [36], Typical analytical methodologies used to assess carbohydrate content are also listed in Table 2. 

The crystallization of a biological macromolecule is realized by manipulation of one or more chemical and thermodynamic variables, such that the solubility of a target molecule in a concentrated solution is reduced, thereby promoting a transition to the solid phase in the form of a well-ordered crystal. In principle, any thermodynamic variable that may directly, or indirectly, affect protein solubility may be used to induce crystallization. Variables that are most often manipulated include macromolecule concentration, ionic strength, identity and concentration of precipitating agents, pH, temperature and small-molecule additives. Together, these variables comprise a vast multi-dimensional chemical phase space that must be systematically explored to discover crystallization conditions. 

Changes in a single experimental parameter can simultaneously influence several aspects of a crystallization experiment. For example, temperature changes affect protein solubility, rates of nucleation and growth, and equilibration of the experimental apparatus. The interaction of parameters makes it difficult to design experiments to isolate individual effects and likewise complicates the interpretation of experimental results. 

Many thermodynamic variables, such as temperature, pH, ionic strength, and other compositions in the solution system, affect protein solubility and compatibility with other macromolecule components of the system. At con-... 

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

Allen et al. (2007) produced puffed snack foods with com starch and pregelatinized waxy starch, WPC and instantized WPC, and protein concentrations of 16%, 32%, and 40% and showed that the air cell size, extru-date expansion ratio, and water solubility index decreased proportionally as protein and com starch levels increased. Protein concentration significantly affected total soluble protein, water absorption index, and water-soluble carbohydrate. A covalent complex between amylase and protein formed in the presence of cornstarch, but protein-protein interactions appeared with the presence of low levels of pregelatinized waxy starch. 

Emulsification is a stabilizing effect of proteins a lowering of the interfacial tension between immiscible components that allow the formation of a protective layer around oil droplets. The inherent properties of proteins or their molecular conformation, denaturation, aggregation, pH solubility, and susceptibility to divalent cations affect their performance in model and commercial emulsion systems. Emulsion capacity profiles of proteins closely resemble protein solubility curves and thus the factors that influence solubility properties (protein composition and structure, methods and conditions of extraction, processing, and storage) or treatments used to modify protein character also influence emulsifying properties. 

Proteins may be fibrous or globular. The structure and polarity of the particular amino acid R groups and their sequence affect the solubility properties and tertiary structure of proteins. Quaternary structure refers to the aggregation of similar protein subunits. 

Several factors affect the selection of the buffer solution, such as the optimum pH the buffer anionic or cationic species (which can interfere in the subsequent purification steps) the pH variation with ionic strength or temperature the buffer reactivity with the proteins in solution the biological activity (e.g. phosphates can inhibit or activate a protein in biological reactions) the interaction of the buffer with other components the buffer permeation in biological membranes the toxicity the light absorption at 280 nm the cost (especially if used in large-scale processes) and the protein solubility. 

The high salt level (1.0 M NaCl) and poor protein solubility evidently affected emulsion formation at pH 1.5 (cf. Figures 5 and 6). At pH 6.7, levels of soluble proteins were variable in... 

The formation of complexes affects both particle-solvent and particle-particle interactions. The solubility of proteins may be increased by their electrostatic complexing with anionic polysaccharides. Formation of titration-complexes may increase protein solubility and inhibit protein precipitation at the lEP. Anionic polysaccharides can act as protective hydrocoUoids inhibiting aggregation and precipitation of like-charged dispersed protein particles, for example, of denatured proteins. This protective action also can increase the stability of protein suspensions and oil-in-water emulsions stabilized by soluble protein-anionic polysaccharide complexes. 

Timasheff and coworkers [5,59-63] were the first to notice that there is a connection between the preferential binding parameter and the aqueous protein solubility. On the basis of their measurements and literature data regarding the preferential binding parameter and the aqueous protein solubility, they concluded that there is a general correlation between these quantities [5,59-63]. Particularly, they concluded that preferential hydration of a protein (/I < 0) is equivalent to a salting-out behavior, i.e. the addition of a cosolvent decreases the protein solubility [5,65]. Thus, the local composition of the components of a mixed solvent is one of the most important factors affecting the aqueous protein solubility [5,40,59-63]. 

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