Precipitation is said to have occurred when a previously soluble protein becomes insoluble following the addition o f a reagent to the protein solution. The choice o f the precipitant used will depend on whether just one protein is being extracted from a solution or whether proteins with differing sizes or solubilities are being fractionated. Changes in pH, temperature, addition o f salts, organic solvents and non-ionic polymers have all been used to precipitate proteins from solution and their method o f precipitation in each case will be discussed briefly. Isoelectric precipitation is most relevant to this thesis and so examples o f proteins precipitated in this manner will be given.
1.7.1. Precipitation by salt addition
Neutral or slightly acidic salts, such as NaCl, Na2S04, NH4(S04)2 , have all been used to precipitate or fractionate proteins. At low concentrations salts may increase protein solubility but at high salt concentrations a process termed ‘salting out’ occurs. KOI, CaCh and NaCl have all been used to solubilise and precipitate certain fibrous proteins, including myosins, coUagens, fibrinogens and keratins (Englard and Seifter, 1990). Salting out occurs because the increased ionic strength o f the solution pulls water molecules away from hydrophobic regions o f the protein leading to increased interaction between hydrophobic patches on the protein surface and effective neutralisation of surface charges. Multivalent anions such as sulphate, phosphate and citrate are preferred in combination with monovalent cations, which do not complex with the protein. The Hofineister series orders anions depending on their precipitating ability with citrate being the most efiScient precipitant and also having the least destabilising effect.
citrate > POl~ > S0^~ > acetate >Cl~ > Br- > N O f > CIO^ > SCN~
The order o f preference for choice o f cations is NH^ > although cost, the density o f the solution compared to the protein and the effect on protein stability should also be considered (Scopes, 1994).
Ammonium sulphate is frequently the salt precipitant o f choice because it is cheap enough to be used on a large scale, it protects proteins from dénaturation in solution and limits bacterial growth, as well as a having a low heat o f solution which reduces the chance o f protein dénaturation when the salt is added (Englard and Seifter, 1990).
1.7.2. Isoelectric protein precipitation
Isoelectric precipitation occurs when the pH o f a protein solution is altered by the addition o f extra acid or base causing the overall charge o f the protein molecule to become zero. The pH at which the protein has no overall charge is termed the isoelectric point and is usually the pH at which the protein precipitates. When the pH is lowered below the protein’s isoelectric point the molecules become protonated and repel, whereas when the pH is raised the molecules lose protons, take on an overall negative charge and repel. At the isoelectric point there is no repulsion because there is no overall charge and the particles tend to aggregate. Isoelectric precipitations tend to depend upon the components present and the behaviour o f a protein in a mixture may be different from its behaviour in a pure solution (Scopes, 1994).
Gentle heat and isoelectric precipitation (pH 4.2) have been described to fractionate whey proteins (Bramaud et a/., 1997) whilst the protein from sunflower seed has been extracted around its isoelectric point, pH 3.6-4.0 (Taha et aL, 1981; Rapheal et al., 1995; Rapheal and Rohani, 1996). Soybean protein has been extensively investigated and precipitated around its isoelectric point between pH 4.0 and 4.8 (LiUford and Wright, 1981; Virkar et a i, 1982).
The examples given above all precipitated in acid conditions, however this is not always the case with some proteins being stable in acidic solutions and precipitating under alkaline conditions. Mineral acids are frequently used for protein precipitation because they are cheap and hydrochloric, sulphuric and phosphoric acids are accepted for human consumption and therefore do not necessarily have to be removed from the final product (BenetaL, 1983).
1.7.3. Precipitation using organic solvents
Solvents such as methanol, ethanol, butanol and acetone have been used to precipitate proteins. All the solvents have both hydrophilic and hydrophobic groups which results in the solvent interacting with the hydrophobic regions o f the proteins and competing with the water to interact with the hydrophilic regions. The solvating power o f the water is decreased as the concentration o f solvent increases, causing molecules to aggregate. Aggregation is increased at the isoelectric point and large proteins require lower concentrations o f solvent to precipitate out. As described in section 1.2. ethanol fractionation is frequently used in the plasma fractionation industry. However there are disadvantages associated with the use o f solvents including a tendency to denature proteins at temperatures above 0°C and the need to design the plant for use with flammable solvents (Englard and Seifter, 1990; Scopes, 1994)
1.7.4. Precipitation using non-ionic polymers
The action o f non-ionic precipitating agents such as PEG has been described previously in section 1.2. along with their use in the plasma fractionation industry. The action o f these polymers as précipitants can be enhanced by alteration o f pH towards the isoelectric point of the protein or with the addition o f divalent metal ions to complex the protein (Scopes, 1994). Ingham (1990) describes the precipitation o f fibronectin with 11 % PEG from a solution o f phosphate-buffered saline. However a concentration o f only 3 % PEG was needed when a solution o f fibronectin and gelatin was being precipitated. Gelatin was found not to precipitate alone under these conditions thus associations between proteins in solution are important during precipitation.
1.7.5. Other less frequently used forms of precipitation
Since not all proteins have the same pH and temperature optima, manipulation o f environmental conditions can selectively denature some proteins whilst leaving others intact. Altering temperature, pH and the presence o f organic solvents, frequently in combination with each other, can result in the loss o f the protein’s tertiary structure and the formation o f free polypeptide chains. These will interact randomly and cause precipitation o f the denatured protein. This happens at extremes o f pH when molecules gain or lose charges which were involved in the stabilisation o f the structure (Scopes, 1994).
Ionic polyelectrolytes, including alginate and carboxymethylcellulose, have also been used in the food industry to precipitate proteins by disrupting the molecular electrostatic interactions. On the addition o f carboxymethylcellulose, precipitation has been found to occur below the protein’s isoelectric point (Bell et al., 1983).
It can be seen that there are a number o f ways o f precipitating proteins but the method selected depends upon the stability o f the protein, its final use and the need to remove contaminating précipitants, the economics o f the project, design o f plant and the separation problem Precipitation is an important feature o f fibre wet spinning which will be discussed next.