Isoelectric point, protein solubility

Just as individual amino acids have isoelectric points, proteins have an overall p/ because of the acidic or basic amino acids they may contain. The enzyme lysozyme, for instance, has a preponderance of basic amino acids and thus has a high isoelectric point (p/= 11.0). Pepsin, however, has a preponderance of acidic amino acids and a low- isoelectric point pi 1.0). Not surprisingly, the solubilities and properties of proteins with different pi s are strongly affected by the pH of the medium. Solubility- is usually lowest at the isoelectric point, where the protein has no net charge, and is higher both above and below the pi, where the protein is charged. 

Glycosylation of the proteins has the potential to influence the interaction of the cell with its environment. The presence of negatively charged pseudaminic acid derivatives on the surface would lower the isoelectric point, influence solubility, and likely increase ionic interactions.15 In C. jejuni flagellin, these may be further controlled by introduction of a basic acetamidino group to the sugar.16... 

Protein precipitation is t rpically observed at high ionic strength, in the presence of organic solvents or close to the isoelectric point, where solubility is low due to the zero net charge of the protein. Presence of precipitates is not always easily observed. T5rpical markers are presence of large and often white particles or flocculates, a turboid appearance, fibrils, or increased viscosity. 

The term euglobulin" as employed in connection with the plasma proteins in disease ordinarily refers to the Howe euglobulin fraction. The same term may be used, however, to indicate proteins precipitated by 0.33-cnturated ammonium sulfate solution, proteins precipitated out of serum by addition of distilled water or by dialysis (proteins insoluble in distilled water near their isoelectric points but soluble in dilute sidt solutions), or proteins precipitated out of serum by lowering the pH, usually by introducing carbon dioxide. Needless to say, much confusion has resulted from these different usages. 

The degree of ionization depends on the pH of the solution. An excess of H+ ions (low pH) retards dissociation of the acidic groups at high pH, the basic groups remain uncharged. At a certain pH, the number of positive and negative charges will be exactly equal. This particular pH is called the isoelectric point (pl). At the isoelectric point, proteins are found to be least soluble and can be precipitated most easily. 

One of the most important methods is salting out with a neutral salt such as ammonium sulfate, sodium sulfate, or magnesium sulfate, since highly concentrated solutions of these salts can be prepared. The salt content of a protein solution is increased stepwise, and after each addition the precipitated protein is centrifuged off. The pH may be varied to allow multiple fractionation (around their isoelectric points proteins become least soluble). Another method is precipitation with organic solvents (alcohol, acetone) according to E. Cohn. This must be carried out under refrigeration (cf. Chapt. IV-9). 

Extract with water at protein isoelectric point Water-soluble ... 

Water-soluble polymers and polyelectrolytes (e.g., polyethylene glycol, polyethylene imine polyacrylic acid) have been used success-hilly in protein precipitations, and there has been some success in affinity precipitations wherein appropriate ligands attached to polymers can couple with the target proteins to enhance their aggregation. Protein precipitation can also be achieved using pH adjustment, since proteins generally exhibit their lowest solubility at their isoelectric point. Temperature variations at constant salt concentration allow for frac tional precipitation of proteins. 

Concentrates are made by extracting water-soluble sugars and other compounds from defatted meals or flours. This is typically a secondary extraction, using acidic ethanol-water in a chain-type or basket-type continuous extractor for processing flakes, or acidic water extraction of flour in vats, followed by spray-drying (8). Acidic polar solvents are used at or near the isoelectric point of the protein to minimize its solubility and loss. The reextracted flakes may then be ground into a flour. Concentrates are more bland than defatted flours, but still contain the fiber components of the kernel. After extraction with acidic ethanol or water, concentrates... 

Figure 5.1 Two-dimensional electrophoresis separates potato proteins based on their isoelectric points and molecular weights. Using a wide-scale pH gradient and large gels, 1000-2000 quantifiable proteins can be separated. The gel stained with SYPRO Rubi shows soluble tuber proteins of potato cultivar Sante.

Emulsifying activity index (EAI) is a measure of the ability of protein to emulsify oil, which depends on solubility, size, charge, and surface activity of the protein molecules. The effect of proteolysis with pronase E on EAI of the modified protein was relatively insignificant (Figure 6) However, deamidation appeared to enhance EAI, especially at pH values more basic than the isoelectric point (pH 4.7). 

Lowers solubility of protein at or near isoelectric point or pi, where the protein is in net unionized form some protein may become denatured under these conditions Heat-sensitive proteins expose hydrophobic domains, which exhibit decreased solubility. This process must be reversible if original conformation is required for protein activity... 

Electrostatic charges due to ionized acidic or basic amino acids influence protein solubility. At extremes of pH, many poorly soluble proteins are dissolved and their molecular structures unfolded due to surplus of similar repelling charges. Gluten proteins have few charged groups and so are poorly soluble in neutral solution (15). Dispersions of other proteins must be adjusted to their isoelectric point or have salt added to optimize cohesion and adhesion. 

Hagenmaier (10) demonstrated that pH had little effect on water absorption of oilseed protein products, but solubility was pH dependent. He suggested that the differing degree of dependence on pH indicates that water absorption and protein solubility are not correlated. Contrastingly, Wolf and Cowan (28) reported the pH-water retention curve of soy proteins to follow the pH-solubility curve. Both solubility and water retention were minimal at the isoelectric point (4.5) and increased as the pH diverged from this point. Hutton and Campbell (20) reported that the effects of pH and temperature on water absorption of soy products paralleled those of solubility for the most part. 

Data in Figure 6 show the effect of varying the pH and sodium chloride concentration on emulsion capacity of peanut protein isolate. Shifting the pH to levels above or below the isoelectric point improved emulsion capacity of peanut protein isolate in O.IM or 0.2M NaCl. Similar trends were noted when distilled water was used as the continuous phase (data not.shown). At the 0.5M NaCl concentration, however, little difference was noted in emulsion capacity at pH 3, 4, or 5 appreciable increases occurred when the pH was raised to 6 and above. At the highest salt concentration (1.OM NaCl), a gradual increase in emulsion capacity occurred when the pH was increased from 3 to 10. An overall suppression in emulsion capacity occurred as salt concentration increased except at pH 5 and 6. These emulsion-capacity curves closely resemble the protein-solubility curves of peanut protein shown in Figure 7... 

---