What are the separation methods in protein? What are their properties or characteristics in protein?

(1) aqueous solution extraction method

Dilute salt and aqueous solution of buffer system are the most commonly used solvents for extracting protein, because they have good stability and high solubility for protein. Usually the dosage is 65,438+0-5 times the volume of raw materials. When extracting, it needs to be stirred evenly to facilitate the dissolution of protein. The extraction temperature depends on the properties of effective components. On the one hand, the solubility of most protein increases with the increase of temperature, so high temperature is beneficial to dissolution and shortens the extraction time. On the other hand, with the increase of temperature, protein will be denatured and inactivated. Therefore, based on this, the extraction of protein and enzyme is generally carried out at low temperature (below 5℃). In order to avoid degradation during the extraction of protein, protease inhibitors (such as diisopropyl fluorophosphate and iodoacetic acid) can be added.

The following focuses on the selection of pH value and salt concentration of the extract.

1, pH value

Protein, the enzyme is an amphoteric electrolyte with isoelectric point, and the pH value of the extract should be selected at the pH values on both sides of the isoelectric point.

Range. When extracting with dilute acid or dilute alkali, it is necessary to prevent the dissociable groups of protein from changing due to peracid or perabase, resulting in irreversible changes of protein conformation. Generally speaking, basic protein is extracted with slightly acidic extract, and acidic protein is extracted with slightly alkaline extract.

2. Salt concentration

Dilution and concentration can promote the dissolution of protein, which is called salt dissolution. At the same time, due to the combination of salt ions and protein, dilute salt solution has the advantage of protecting protein invariance. Therefore, a small amount of neutral salt, such as NaCl, is added to the extract, and the concentration is generally 0. 1.5 mol L, and the buffer solution is usually 0.02-0.05M of phosphate and carbonate.

(2) organic solvent extraction method

Some protein and enzymes that are closely bound to lipids or contain more nonpolar side chains in their molecules are insoluble in water, dilute salt solution, dilute acid or dilute alkali. Organic solvents such as ethanol, acetone and butanol can be used. They have certain hydrophilicity and strong lipophilicity and are ideal extraction solutions for lipoproteins. However, they must be operated at low temperature. Butanol extraction method is especially beneficial to extract some protein and enzymes closely combined with lipids. First of all, butanol has strong lipophilicity, especially for dissolution. Secondly, butanol is hydrophilic, and it will not cause denaturation and inactivation of the enzyme within the solubility range (10% and 6.6% at 40℃). In addition, the butanol extraction method has a wide pH and temperature range, and is also suitable for animals, plants and microbial materials.

Second, the separation and purification of protein

There are many methods for the separation and purification of protein, mainly including:

(a) according to the protein solubility of different separation methods.

1, salting out in protein

Neutral salts have a significant effect on the solubility of protein. Generally, at low salt concentration, with the increase of salt concentration, the solubility of protein increases, which is called salt dissolution. When the salt concentration continues to increase, the solubility of protein decreases to varying degrees and precipitates one after another. This phenomenon is called salting out. When a large amount of salt is added to protein solution, high concentration of salt ions (such as SO4 and NH4 of ammonium sulfate) have strong hydration force, which can grasp the hydration layer of protein molecules and make it "dehydrated", so protein colloidal particles coagulate and precipitate. When salting out, the solution pH is better at the isoelectric point of protein. Due to various protein molecular particles,

The factors affecting salting-out are: (1) temperature: protein is sensitive to temperature, so it can generally be operated at room temperature. Generally speaking, the solubility of protein will decrease at low temperature, but the solubility of some protein (such as hemoglobin, myoglobin and albumin) is lower than 0 degree at higher temperature (25 degrees). It's easier to salt out. (2)pH value: Most protein has the lowest solubility in concentrated salt solution at isoelectric point. (3) protein concentration: When the concentration of protein is high, protein to be separated often precipitates with other places in protein (* * * precipitation phenomenon). Therefore, before salting out, the serum should be diluted with the same amount of normal saline to make the protein content reach 2.5-3.0%.

The neutral salts commonly used in protein salting-out mainly include ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride and sodium phosphate.

The most widely used ammonium sulfate has the advantages of low temperature coefficient and high solubility (the saturated solution is 4. 1M at 25℃, that is, 767 g/l; The saturated solubility at 0℃ is 3.9M, that is, 676 g/L). In this solubility range, many protein and enzymes can be salted out. In addition, the staged salting-out effect of ammonium sulfate is better than other salts, and it is not easy to cause protein denaturation. The pH value of ammonium sulfate solution is usually between 4.5 and 5.5. When salting out with other pH values, it needs to be adjusted with sulfuric acid or ammonia water.

Protein needs to remove salt from protein after salting out and precipitation separation. The usual method is dialysis, that is, protein solution is put into a bag for analysis (usually cellophane), dialyzed with buffer, and the buffer is constantly changed. Because dialysis takes a long time, it is best to do it at low temperature. In addition, it can also be used to remove salt by passing glucose gel G-25 or G-50 through a column, which takes a short time.

2, isoelectric point precipitation method

When protein is in an electrostatic state, the electrostatic repulsion between particles is the smallest, so the solubility is the smallest. The isoelectric points of various protein are different. It can be precipitated by adjusting the pH value of the solution to a protein isoelectric point, but this method is rarely used alone and can be combined with salting-out method.

3. Low temperature organic solvent precipitation method

The use of water-miscible organic solvents, such as methanol, ethanol or acetone, can reduce the solubility of most protein and precipitate it. The resolution of this method is higher than that of salting-out method, but protein is easy to denature, so it should be carried out at low temperature.

(2) According to the different separation methods of protein molecules.

1, dialysis and ultrafiltration

Dialysis is the use of semi-permeable membrane to separate protein with different molecular sizes.

Ultrafiltration uses high pressure or centrifugal force to force small molecules of water and other solutes to pass through a semi-permeable membrane, while protein remains on the membrane. Filter membranes with different pore sizes can be selected to intercept protein with different molecular weights.

2. Gel filtration method

Also known as molecular exclusion chromatography or molecular sieve chromatography, it is one of the most effective methods to separate protein mixture according to its molecular size. The most commonly used packing material in the column is glucose gel (Sephadex

Ged) and agarose gel.

(3) According to the charged nature of protein.

Protein has different charged properties and charges in different pH environments, so it can be separated.

1, electrophoresis

Under the same pH condition, due to different molecular weights and charges, various protein have different mobility in the electric field, so they can be separated. It is worth noting that isoelectric focusing electrophoresis uses an amphoteric electrolyte as a carrier. During electrophoresis, the amphoteric electrolyte forms a gradually increasing pH gradient from the positive electrode to the negative electrode. When protein with a certain charge swims in it, the pH position at its isoelectric point stops. This method can be used to analyze and prepare various protein.

2. Ion exchange chromatography

Ion exchangers include cation exchangers (such as carboxymethyl cellulose; CM- cellulose) and anion exchanger (diethylaminoethyl cellulose; DEAE？ script

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LANG = " ZH-CN " & gt； Cellulose), when the separated protein solution flows through the ion exchange chromatography column, the protein with the opposite charge to the ion exchanger is adsorbed on the ion exchanger, and then the adsorbed protein is eluted by changing the pH or ionic strength (see the chapter on chromatography technology for details).

(4) Affinity chromatography, a separation method based on ligand specificity.

affinity chromatography

Chromatography is a very effective method to separate protein. It usually takes only one step to separate a certain protein to be purified from a very complicated protein mixture. And the purity is very high. This method is based on the fact that some protein can specifically bind to another molecule called ligand instead of * * *. The basic principle is that protein exists in tissues or cells in the form of complex mixtures, and each type of cells contains thousands of different protein, so protein is isolated and purified.

Characterization is an important part of biochemistry. So far, there is no single or ready-made method to extract any protein from complex mixed protein, so several methods are often used in combination.

cell division

1. High-speed tissue mashing: prepare the material into a thin paste, put it into a cylinder with a volume of about 1/3, cover the cylinder head tightly, first set the governor to the slowest position, then turn on the switch, and gradually accelerate to the required speed. This method is suitable for internal organs of animals, fleshy seeds of plants, etc.

2. Homogenize with a glass homogenizer: firstly, put the cut tissue into a test tube, then put it into a grinding rod to grind it back and forth, and move the grinding cells up and down. This method has a higher degree of cell fragmentation than the high-speed tissue masher, and is suitable for a small number of animal organs and tissues.

3. Ultrasonic wave treatment: the cell suspension is treated by ultrasonic wave with a certain power, so that the cells vibrate violently and rupture. This method is most suitable for microbial materials. For the preparation of various enzymes by Escherichia coli, the concentration of 50- 100 mg bacteria /ml is often selected, and the frequency is 1KG to 10KG.

4. Repeated freezing and thawing method: freeze the cells below -20℃, melt at room temperature, and repeat several times. Due to the formation of ice particles in cells and the increase of salt concentration in residual cell fluid, they expand and destroy the cell structure.

5. Chemotherapy: Some animal cells, such as tumor cells, can be destroyed by cell membranes such as sodium dodecyl sulfate (SDS) and sodium deoxycholate, and the bacterial cell wall is thick, so lysozyme can be used for better treatment.

No matter which method is used to break tissues and cells, protein or nucleic acid hydrolase in cells will be released into the solution, leading to the biodegradation of macromolecules and the reduction of natural products. Adding diisopropyl fluorophosphate (DFP) can inhibit or slow down autolysis. Adding iodoacetic acid can inhibit the activity of proteolytic enzymes whose active center needs hydrophobic groups, and adding benzyl sulfonyl fluoride (PMSF) can also remove the activity of proteolytic chips, but not all of them. By choosing pH, temperature and ionic strength, these conditions should be suitable for the extraction of the target substance.

Concentration, drying and preservation

I. Sample concentration

In the process of preparing biological macromolecules, the samples become very dilute due to column purification, so it is often necessary to concentrate them for preservation and identification. Common concentration methods are:

1, decompression, heating, evaporation and concentration

Reduce the boiling point of liquid by reducing the surface pressure. The higher the vacuum degree of decompression, the lower the boiling point of liquid and the faster the evaporation. This method is suitable for the concentration of some thermally unstable biological macromolecules.

2. Evaporation concentration of airflow

The flow of air can accelerate the evaporation of liquid and make it spread into a thin layer of solution, and the surface constantly passes through the airflow; Or put the biomacromolecule solution into a dialysis bag and put it in a cold room, and blow it with an electric fan to prevent the solvent outside the permeable membrane from evaporating, so as to achieve the purpose of concentration. This method has a slow concentration speed and is not suitable for the concentration of a large number of solutions.

3. Solidification method

Biomacromolecules form ice at low temperature, and salts and biomacromolecules stay in the liquid phase without entering the ice. In operation, the solution to be concentrated is cooled into a solid, and then slowly melted, and most of the solvent is removed by using the difference of melting points between the solvent and solute. For example, when the salt solution of protein and enzyme is concentrated in this way, pure ice crystals without protein and enzyme float on the liquid surface, while protein and enzyme are concentrated in the lower solution, and the upper ice is removed, so that protein and enzyme can be obtained.

4. Absorption method

The solution molecules in the solution are directly removed by the absorbent to concentrate it. The absorbent used must not react chemically with the solution, adsorb biological macromolecules and be easily separated from the solution. Commonly used adsorbents include polyethylene glycol, polyvinylpyrrolidone, sucrose and gel. When using polyethylene glycol absorbent, the biomacromolecule solution should be put into a semipermeable membrane bag first, and then polyethylene glycol should be covered at 4 degrees, and the solvent in the bag will be quickly absorbed by polyethylene glycol. When polyethylene glycol is saturated with water, it should be replaced with a new one.

5. Ultrafiltration method

Ultrafiltration is a method of selectively filtering various solute molecules in solution by using special membranes. When no liquid passes through the membrane under a certain pressure (nitrogen pressure or vacuum pump pressure), solvents and small molecules penetrate, and macromolecules are blocked and intercepted. This is a new method developed in recent years, which is most suitable for the concentration or desalination of biological macromolecules, especially protein and enzymes. It has the advantages of low cost, convenient operation and mild conditions, and can keep the activity of biological macromolecules well. The recovery rate is high. The key to the application of ultrafiltration is the choice of membrane. Different types and specifications of membranes have different parameters, such as water flow rate and molecular weight cut-off value (that is, the minimum molecular weight of molecules that can be intercepted by membranes in general), which must be selected according to the needs of work. In addition, the form of ultrafiltration device, composition and properties of solute, solution concentration, etc. All of them have certain influence on the ultrafiltration effect. Diaflo

Molecular weight cutoff value of ultrafiltration membrane:

Membrane name molecular weight cut-off value large average pore size

XM-30030000 140

XM-200 100，00055

XM-5050，00030

PM-30 30，00022

UM-2020，000 18

PM- 10 10，000 15

UM-2 1，000 12

UM05500 10

Hollow fiber tubes are made of the above-mentioned ultrafiltration membranes, and many of these tubes are gathered into bundles. The two ends of the tube are connected with a buffer solution with low ionic strength, so that the buffer solution can continuously flow in the tube. Then the fiber tube is immersed in protein solution to be dialyzed. When the buffer solution flows through the fiber tube, small molecules easily diffuse through the membrane, while large molecules cannot. This is the fiber filtration analysis method, which shortens the dialysis time by 65,438+00 due to the increase of dialysis area.

Second, drying.

In order to prevent deterioration and facilitate storage, products prepared from biological macromolecules often need drying, and the most commonly used methods are freeze-drying and vacuum drying. Vacuum drying is suitable for the drying and preservation of substances that are not resistant to high temperature and easy to oxidize. The whole device includes dryer, condenser and vacuum drying principle, and the temperature coefficient is increased at the same time. At the same pressure, the pressure of water vapor decreases with the decrease of temperature, so at low temperature and low pressure, ice can easily sublimate into gas. In operation, the liquid to be dried is usually frozen below freezing point to make it solid, and then the solvent is converted into gas at low temperature and low pressure to be removed. The dried product is loose, has good solubility and natural structure, and is suitable for drying and preservation of various biological macromolecules.

Third, storage

The stability of biological macromolecules is closely related to preservation methods. The dried product is generally stable, and its activity will not change obviously for several days or even years at low temperature. Storage requirements are very simple, as long as the dried samples are sealed in a dryer (filled with desiccant) and stored in a refrigerator at 0-4 degrees. When storing in liquid state, the following points should be noted.

1, the sample should not be too dilute, and it must be concentrated to a certain concentration before it can be packaged and stored. Too thin samples are easy to denature biological macromolecules.

2. Generally, preservatives and stabilizers need to be added. Commonly used preservatives are toluene, benzoic acid, chloroform, thymol and so on. Protein and enzyme are usually stabilized by ammonium sulfate paste, sucrose, glycerol, etc. For example, enzymes can also add substrates and coenzymes to improve their stability. In addition, solutions such as calcium, zinc and boric acid also have certain protective effects on some enzymes. Nucleic acid macromolecules are usually stored in standard buffer of sodium chloride or sodium citrate.

3, the storage temperature is low, most of them are stored in the refrigerator at about 0 degrees, and some are lower, depending on different substances.