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3. Isoelectric precipitation is used for the precipitation of amino acids, proteins and other amphoteric substances. However, this method is used alone and is often used in combination with other methods.
4. Nonionic polymer precipitation methods are used to separate biomacromolecules.
5. The salt complex precipitate is formed for the precipitation of a variety of compounds, particularly small molecules.
6. The salting out method is mostly used for the separation and purification of various proteins and enzymes.
The first section of salting out
In general, all solid solutes can be precipitated by adding a neutral salt to the solution. This process is called salting out. In biochemical preparation, many substances can be precipitated by salting out, such as proteins, peptides, polysaccharides, nucleic acids, etc. Among them, protein precipitation is the most common, especially in the crude extraction stage.
The salting out method is divided into two categories. The first type is called Ks segmental salting out method. It is realized by changing the ionic strength at a certain pH and temperature for the early crude extract; the second is called b-segmented salting-out method. It is achieved by varying the pH and temperature at a certain ionic strength for further separation, purification and crystallization.
one. Several factors affecting salting out Protein concentration high concentration protein solution can save the amount of salt, but the b and Ks constants of many proteins are very close. If the protein concentration is too high, serious coprecipitation will occur; salting out in low concentration protein solution, the amount of salt used More, and the co-precipitation is less, so you need to choose between the two. For the step separation and purification, it is better to choose a rare protein solution and add a little neutral salt to minimize the coprecipitation. It is generally considered that the protein concentration of 2.5%-3.0% is moderate.
2. Ion Strength and Type In general, the greater the ionic strength, the lower the solubility of the protein. When performing the separation, it is generally carried out sequentially from a low ionic strength to a high ionic strength. After each component is salted out, it is collected by filtration or freeze centrifugation, and then the neutral salt saturation is gradually increased in the solution to cause another protein component to be salted out.
Ion species also have a certain effect on protein solubility. Ions with small ionic radius and high charge have a strong influence on salting out. The effect of ions with large ionic radius and low charge is weak. The following is the arrangement of salting out ability of several salts. Order: potassium phosphate > sodium sulfate > ammonium phosphate > sodium citrate > magnesium sulfate.
3. PH value Generally speaking, the more the net charge of the protein, the greater the solubility, the less the net charge, the smaller the solubility, and the less the protein solubility at the isoelectric point. In order to improve the efficiency of salting out, the pH of the solution is adjusted to the isoelectric point of the target protein. However, it must be noted that the isoelectric point of the protein in water or dilute salt solution is different from that measured under high salt concentration. The pH value of the solution should be adjusted according to the actual situation to achieve the best salting out effect.
4. Temperature In low ionic strength or pure water, protein solubility increases with increasing temperature within a certain range. However, at high concentrations, the solubility of proteins, enzymes, and peptides decreases with increasing temperature. In general, proteins have no special requirements for salting-out temperature and can be carried out at room temperature. Only certain temperature-sensitive enzymes are required to be carried out at 0-4 °C.
two. Use of ammonium sulfate
Ammonium sulphate often contains a small amount of heavy metal ions, which is sensitive to protein sulfhydryl groups. It must be treated with H2S before use: ammonium sulphate is made into a concentrated solution, saturated with H2S, placed overnight, and heavy metal ions are removed by filter paper, concentrated and crystallized, 100 ° C Use after drying. In addition, the high concentration ammonium sulfate solution is generally acidic (pH = about 5.0), and it is also necessary to adjust to the desired pH with ammonia water or sulfuric acid before use.
There are several methods for adding ammonium sulfate: 1) adding a solid salt method for requiring a higher saturation without increasing the volume of the solution; 2) adding a saturated solution method for requiring a low saturation without the original solution volume Large condition; 3) Dialysis balance method First, the sample of salting out is placed in a dialysis bag, and then immersed in saturated ammonium sulfate for dialysis, and the saturation of ammonium sulfate in the dialysis bag is gradually increased. After reaching the set concentration, the target protein is precipitated. Stop dialysis. The advantage of this method is that the change of ammonium sulfate concentration is continuous, the salting out effect is good, but the procedure is cumbersome, and it is necessary to continuously measure the saturation, so it is mostly used for crystallization, and other cases are rare.
When using solid ammonium sulfate: 1) Must pay attention to the temperature specified in the saturation table, generally 0 ° C or room temperature, the volume change after adding solid salt has been considered in the table; 2) in the segmentation salting, should be considered The change in protein concentration after each segmentation. For a protein such as secondary dialysis, in general, the first salting out separation range (saturation range) is relatively wide, and the second separation range is narrow. 3) After salting out, it is usually placed for half an hour to one hour, and then filtered or centrifuged after the precipitation is completed. Filtration is mostly used for high-concentration ammonium sulfate solution, because in this case, the density of ammonium sulfate is large, and if centrifugation requires high centrifugal speed and long-time centrifugation operation, it takes time and energy. Centrifugation is mostly used for low concentration ammonium sulfate solution.
Section 2 Organic Solvent Precipitation
The precipitation mechanism of the organic solvent is to lower the dielectric constant of water, resulting in dehydration of the biomacromolecules having a surface water layer, aggregation with each other, and finally precipitation. The advantages of this method are: 1) the resolution is higher than the salting out method, that is, the protein or other solvent is precipitated only at a relatively narrow organic solvent concentration; 2) the precipitation is not desalted, the filtration is easy; 3) the application ratio in biochemical preparation The salting out method is extensive. The disadvantage is that the biologically active macromolecule is liable to cause degeneration and deactivation, and the operation is required to be carried out at a low temperature. In general, organic solvent precipitation of proteins and enzymes is not as common as salting out.
The choice of organic solvent is firstly miscible with water. The more organic solvents used are ethanol, methanol, acetone, as well as dimethylformamide, dimethyl sulfoxide, acetonitrile and 2-methyl-2,4 pentane. Glycol and the like.
There are many factors affecting the precipitation effect of organic solvents: 1) The temperature at low temperature can maintain the activity of biomacromolecules, while reducing its solubility and improving the extraction efficiency; 2) the sample concentration and pH are basically the same as those in the salting out method; 3) metal Some polyvalent cations such as Zn2+ and Ca2+ can form complexes with proteins in an anionic state at a certain pH. The solubility of this complex in water or organic solvents is greatly reduced, and does not affect the biological activity of the protein. 4) The ionic strength salt concentration is too large or too low, which has an adverse effect on the separation. For proteins and polysaccharides, the salt concentration is not more than 5%, and the amount of ethanol used is not more than twice the volume.
Section III Other precipitation methods
one. Isoelectric precipitation method has the lowest solubility when the net charge on the ampholyte molecule is zero, and different ampholytes have different isoelectric points, which can be separated based on this. For example, when industrially producing insulin, the alkaline protein is first adjusted in the crude extract by adjusting pH 8.0, and the acidic protein is removed by adjusting pH 3.0.
When using the isoelectric point to remove the protein, it is necessary to understand the stability of the preparation to the acid and base, otherwise blind use is very dangerous. After a lot of proteins are combined with metal ions, the isoelectric point will shift. Therefore, when the metal ions are contained in the solution, care must be taken to adjust the pH. The isoelectric point method is often used in combination with salting out, organic solvent precipitation or other precipitation methods to increase its precipitation capacity.
two. Formation of salt complex precipitation method Metal complex salt method Many organic substances, including proteins, are negatively charged in an alkaline solution and can form a precipitate with metal ions. According to the interaction mechanism between organic substances and them, they can be divided into nitrogen-containing compounds such as carboxylic acids, amines and heterocyclic rings, such as copper-zinc cadmium; carboxylic acid-containing nitrogen-containing compounds such as magnesium hydride; thiol-hydrogen-based compounds. Classes such as mercury, silver and lead. An important property of protein-metal ion complexes is that their solubility is very sensitive to the dielectric constant of the solution. By adjusting the dielectric constant of the aqueous solution (such as adding an organic solvent), a variety of proteins can be precipitated.
2. Organic salt method Nitrogen-containing organic acids such as picric acid, piculic acid, citric acid and the like can form a complex with an alkaline functional group of an organic molecule to precipitate and precipitate. However, this method often has an irreversible precipitation reaction, so when it is used to prepare a protein, it is necessary to adopt a milder condition, and sometimes a certain stabilizer is added.
3. Inorganic composite salt methods such as phosphotungstate, phosphomolybdate, and the like.
The above salt complexes have a very low solubility and are easily precipitated. If the precipitate is a metal complex salt, it can be removed by changing the metal into a sulfide by H2S. If it is an organic acid salt or a phosphotungstate, the inorganic acid is added and extracted with diethyl ether, and the organic acid and the phosphotungstic acid are transferred into the ether. Removed or removed by ion exchange. It is worth noting that such methods often cause irreversible precipitation of proteins, and care must be taken when applying them.
three. The principle of selective denaturation precipitation is to use biological macromolecules such as proteins, enzymes and nucleic acids to be sensitive to certain physical or chemical factors, and selectively denature and precipitate to achieve the purpose of separation and purification.
The method can be divided into: 1) using a surfactant (trichloroacetic acid) or an organic solvent to cause denaturation; 2) utilizing heat instability, heating to destroy certain components while preserving other components; 3) acid Alkali denaturation.
four. Nonionic polymer precipitation nonionic polymer is an important type of precipitant developed in the 1960s. It was first used to purify immunoglobulins, precipitate some bacteria and viruses, and has been widely used in nucleic acid and enzyme separation in recent years. Purification. Such nonionic polymers include polyethylene glycols of different molecular weights, NPEO, dextran, sodium dextran and the like, of which polyethylene glycol is the most widely used.
There are two methods for precipitating biomacromolecules and microparticles with nonionic polymers: 1) Two liquid-soluble two-phase systems composed of two water-soluble nonionic polymers are used, which are not distributed in equal amounts, resulting in separation. This method is based on the different surface structures of different biomolecules and has different partition coefficients. The effect of ionic strength, pH value and temperature is added to expand the separation effect. 2) A water-soluble nonionic polymer is selected so that the biomacromolecules are precipitated in the same liquid phase due to being repelled and mutually agglomerated. When the method is operated, the large suspended particles are first removed by centrifugation, the pH value and the temperature of the solution are adjusted to moderate, and then the neutral salt and the polymer are added to a certain concentration, and stored for a period of time to form a precipitate.
Cell membrane protein extraction method--protein precipitation method
1. Thermal denaturation and acid-base denaturation precipitation methods are used to selectively remove certain heterogeneous proteins that are not heat-resistant and are susceptible to denaturation at a certain pH. 2. Organic solvent precipitation is widely used for the separation and purification of biological small molecules, polysaccharides and nucleic acid products, and sometimes for protein precipitation.