1. Heat denaturation and acid-base denaturation precipitation methods are used to selectively remove certain hetero-proteins that are not heat-resistant and are easily degraded at a certain pH. 2. Organic solvent precipitation method is widely used for the separation and purification of biological small molecules, polysaccharides and nucleic acid products, and sometimes used for protein precipitation.
3. Isoelectric precipitation method for the precipitation of amino acids, proteins and other amphoteric substances. However, this method is used on its own and is often used in combination with other methods.
4. Non-ionic polymer precipitation method for the separation of biological macromolecules.
5. The formation of salt complex precipitates for the precipitation of a variety of compounds, particularly small molecules.
6. Salting method is used for the separation and purification of various proteins and enzymes.
Section 1 Salting out
In general, all solid solutes can be precipitated by adding neutral salts to the solution. This process is called salting out. In biochemical preparation, many substances can be precipitated and separated by salting out methods, such as proteins, peptides, polysaccharides, and nucleic acids, among which protein precipitation is the most common, especially in the crude extraction stage.
The salting out method is divided into two types. The first type is called Ks divided salting method. It is achieved by changing the ionic strength at a certain pH and temperature. It is used for early crude extracts; the second method is called b-segmentation salting out method. It is achieved by changing the pH and temperature at a certain ionic strength for further separation, purification and crystallization at a later stage.
I. Several factors that affect salt precipitation
Protein concentration
High concentration of 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, a serious coprecipitation will occur; in the low concentration of protein solution salting, the amount of salt used is more However, co-precipitation is relatively small, so it is necessary to choose between the two. When used in step-by-step separation and purification, it is preferable to choose a slightly diluted protein solution and add a little neutral salt to minimize coprecipitation. It is generally believed that the protein concentration of 2.5%-3.0% is relatively moderate.
2. Ionic strength and type
In general, the higher the ionic strength, the lower the solubility of the protein. At the time of separation, it is generally performed sequentially from low ionic strength to high ionic strength. After each component is salted out, it is collected by filtration or refrigerated centrifugation, and then the neutral salt saturation is gradually increased in the solution to salt out another protein component.
Ionic species also have a certain influence on protein solubility. Ion radii are small and highly charged ions have strong influence on salting out. Ion radii are large and low-charged ions have weaker influence. 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
In general, the greater the net charge of a protein, the greater the solubility, the lower the net charge, and the less the solubility of the protein at the isoelectric point. To increase the efficiency of salting out, the pH of the solution is often adjusted to the isoelectric point of the target protein. However, it must be noted that the isoelectric point of the protein in water or diluted salt solution is different from that measured under high salt concentration. The pH of the solution must be adjusted according to the actual conditions to achieve the best salting-out effect.
4. Temperature
In low ionic strength or pure water, protein solubility increases with temperature in a certain range. However, at high concentrations, the solubility of proteins, enzymes, and peptides decreases with increasing temperature. In general, the protein has no special requirements for the temperature of salting out, and can be performed at room temperature. Only certain temperature-sensitive enzymes are required to be performed at 0-4°C.
II. Use of ammonium sulfate
Ammonium sulphate often contains a small amount of heavy metal ions, and has a sensitive effect on the sulfhydryl groups of the protein. Before use, it must be treated with H2S: Ammonium sulphate should be mixed into a concentrated solution, which should be saturated with H2S and left overnight. The heavy metal ions should be removed with filter paper, and the crystals should be concentrated at 100°C. Use after drying. In addition, the high concentration of ammonium sulfate solution is generally acidic (PH = 5.0 or so), and it needs to be adjusted to the desired pH with ammonia or sulfuric acid before use.
Ammonium sulphate can be added in the following ways: 1) The solid salt method is used for the case where higher saturation is required without increasing the volume of the solution; 2) The saturated solution method is used for the requirement that the saturation is not high and the original solution volume is not Large condition; 3) Dialysis balance method The salt precipitation sample is first packed in a dialysis bag and then immersed in saturated ammonium sulfate for dialysis. 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 there is continuity in the change of ammonium sulfate concentration, and the salting-out effect is good, but the procedure is cumbersome and the saturation needs to be constantly measured, so it is mostly used for crystallization, and other situations 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, after the addition of solid salt volume change has been considered in the table; 2) segmentation salting, should be considered Changes in protein concentration after each segmentation. If a protein is subjected to secondary dialysis, generally, the first salting-out separation range (saturation range) is relatively wide, and the second separation range is narrower. 3) After salting out, it is usually kept for half an hour to one hour, and it will be filtered or centrifuged after the precipitation is complete. The filter is mostly used for high-concentration ammonium sulfate solution because in this case, ammonium sulfate has a relatively high density. If the centrifugal method requires a high centrifugal speed and a long-time centrifugal operation, it consumes time and energy. Centrifugation is mostly used for low concentrations of ammonium sulfate solution.
Section 2 Organic solvent precipitation method
The mechanism of precipitation of organic solvents is to reduce the dielectric constant of water, leading to the dehydration of biological macromolecules with surface water layers, mutual aggregation, and finally precipitation. The advantages of this method lie in: 1) higher resolving power than salting-out method, that is, protein or other solvent is only precipitated in a relatively narrow organic solvent concentration; 2) precipitation is not desalted and filtration is relatively easy; 3) application ratio in biochemical preparation Salting method is extensive. The disadvantage is that the biologically active macromolecules are prone to denature deactivation, and the operation is required to be performed at a low temperature. In general, organic solvent precipitation of proteins and enzymes is not as common as salting-out.
The first choice of organic solvent is water-miscible. The most commonly used organic solvents are ethanol, methanol, acetone, dimethylformamide, dimethyl sulfoxide, acetonitrile and 2-methyl-2,4pentane. Diols, etc.
There are many factors that affect the precipitation effect of organic solvents: 1) Temperature and low temperature can maintain the activity of biological macromolecules, at the same time reduce its solubility and increase the extraction efficiency; 2) The concentration and pH of the sample are basically the same as those in the salting out method; 3) Metals Ions Some polyvalent cations such as Zn2+ and Ca2+ can form complexes with proteins in the anionic state at a certain pH. The solubility of this complex in water or organic solvents is greatly reduced without affecting the biological activity of the protein. 4) Ionic strength The salt concentration is too large or too low to have an adverse effect on the separation. For the protein and polysaccharide, the salt concentration is not more than 5%, and the amount of ethanol used should not exceed two volumes.
Section III Other precipitation methods
I. Isoelectric precipitation method
When the net charge on the amphiphilic electrolyte is zero, the solubility is the lowest, and the different ampholytes have different isoelectric points, on the basis of which the separation can be carried out. For industrial production of insulin, PH8.0 is first used to remove basic proteins in the crude extract, and PH3.0 is then added to remove acidic proteins.
When using the isoelectric point to remove the miscible protein, it is necessary to understand the stability of the preparation against acid and alkali, otherwise blind use is very dangerous. After many proteins and metal ions are combined, the isoelectric point shifts. Therefore, when the solution contains metal ions, the pH must be adjusted. The isoelectric point method is often used in combination with a salting out method, an organic solvent precipitation method, or other precipitation methods to increase its precipitation ability.
II. Formation of Salt Complex Precipitation
1. Metal composite salt method
Many organic substances, including proteins, are negatively charged in alkaline solutions and precipitate with metal ions. According to the mechanism of interaction between organic substances and them, they can be divided into nitrogen compounds such as carboxylic acids, amines and heterocyclic rings, such as copper, zinc and cadmium; nitrogen-containing compounds of prolinecarboxylic acids, such as lead magnesium; hydrogenphilic compounds. Classes, such as mercury-silver lead. The important property of protein-metal ion complexes is that their solubility is very sensitive to the dielectric constant of the solution. Adjusting the dielectric constant of the aqueous solution (eg adding organic solvents) can precipitate a variety of proteins.
2. Organic salt method
Nitrogen-containing organic acids such as picric acid, ketoprotic acid, and decanoic acid can form complexes with basic functional groups of organic molecules and precipitate out. However, this method often occurs irreversible precipitation reaction, so when the preparation of protein, the need to use mild conditions, and sometimes also need to add a certain amount of stabilizer.
3. Inorganic complex salt method
Such as phosphotungstate, phosphomolybdate and so on.
The above salt complexes have very low solubility and are easily precipitated. If the precipitate is a metal complex salt, it can be removed by H2S so that the metal becomes sulfide. If it is an organic acid salt or phosphotungstate, the inorganic acid is added and extracted with ether. The organic acid and phosphotungstic acid are transferred into ether. Removed or removed by ion exchange. It is worth noting that such methods often cause irreversible precipitation of proteins and must be used with caution.
III. Selective degeneration precipitation
The principle is to use proteins, enzymes, nucleic acids and other biological macromolecules to have different sensitivities to certain physical or chemical factors, and selectively denature them to achieve separation and purification.
This method can be divided into: 1) using a surfactant (trichloroacetic acid) or an organic solvent to cause denaturation; 2) using thermal instability, heating to destroy certain components while preserving other components; 3) acids Alkaline denaturation.
IV. Non-ionic polymer precipitation method
Nonionic polymers are an important class of precipitants developed in the 1960s. They were used to purify immunoglobulins and precipitate some bacteria and viruses. They have been widely used in the separation and purification of nucleic acids and enzymes in recent years. Such nonionic polymers include polyethylene glycol, NPEO, dextran, sodium dextran sulfate, and the like having different molecular weights, of which polyethylene glycol is the most widely used.
There are generally two methods for precipitating biological macromolecules and microparticles with non-ionic polymers: 1) Two liquid-liquid two-phase systems composed of two water-soluble non-ionic polymers are used, which are separated by unequal distribution. This method is based on different surface structures of different biomolecules, with different partition coefficients. In addition, the effects of ionic strength, pH, and temperature are added to increase the separation effect. 2) A water-soluble nonionic polymer is selected so that the biological macromolecules precipitate in the same liquid phase due to mutual repulsion. When the method is operated, the large suspended particles are first centrifuged to remove the pH of the solution and the temperature is adjusted to a moderate degree, and then neutral salt and polymer are added to a certain concentration, and the precipitate is formed by cold storage for a period of time.