Material Selection and Pretreatment

Understanding life phenomena at the molecular level, based on protein structure and function, has become the main direction of modern biology. To study proteins, highly purified and biologically active target substances must first be obtained. Protein preparation involves knowledge from physics, chemistry, and biology, but the basic principles are nothing more than two aspects. One is to utilize the difference in distribution rates of several components in a mixture, distributing them into two or more phases that can be separated by mechanical methods, such as salting out, organic solvent extraction, chromatography, and crystallization; the other is to place the mixture in a single phase, and achieve separation by distributing components into different regions through the action of a physical force field, such as electrophoresis, ultracentrifugation, and ultrafiltration. In the application of all these methods, attention must be paid to preserving the integrity of biomacromolecules, preventing the loss of biological activity of the extracted substance due to acid, alkali, high temperature, or violent mechanical action. Protein preparation is generally divided into the following four stages: material selection and pretreatment, cell disruption and organelle separation, extraction and purification, concentration, drying, and preservation.S

Microorganisms, plants, and animals can all serve as raw materials for protein preparation. The choice of material is primarily determined by the experimental objective. For microorganisms, their growth phase should be noted; during the logarithmic growth phase, the content of enzymes and nucleic acids is higher, allowing for high yields. When using microorganisms as material, there are two situations: (1) utilizing metabolic products and extracellular enzymes secreted by microbial cells into the culture medium; (2) utilizing biochemical substances contained within microbial cells, such as proteins, nucleic acids, and intracellular enzymes. Plant materials must be de-shelled and defatted, and it should be noted that the amount of biomacromolecules contained varies greatly with different plant varieties and growth and development conditions, and is also closely related to seasonality. For animal tissues, organ tissues rich in active ingredients must be selected as raw materials, and subjected to crushing, defatting, and other treatments first. Additionally, if pretreated materials are not used immediately for experiments, they should be stored frozen. For easily decomposable biomacromolecules, fresh materials should be used for preparation.S

Protein Separation and Purification

I. Protein (including enzyme) Extraction

Most proteins are soluble in water, dilute salt, dilute acid, or alkaline solutions. A few proteins combined with lipids are soluble in organic solvents such as ethanol, acetone, and butanol. Therefore, different solvents can be used to extract, separate, and purify proteins and enzymes.S

(I) Aqueous Solution Extraction Method

Dilute salt and buffered aqueous solutions provide good protein stability and high solubility, making them the most commonly used solvents for protein extraction. The usual amount is 1-5 times the volume of the raw material. Uniform stirring is required during extraction to facilitate protein dissolution. The extraction temperature depends on the nature of the active ingredient. On one hand, the solubility of most proteins increases with temperature, so higher temperatures facilitate dissolution and shorten extraction time. On the other hand, increased temperature can cause protein denaturation and inactivation. Therefore, considering this point, protein and enzyme extraction is generally performed at low temperatures (below 5 degrees Celsius). To prevent degradation during protein extraction, protease inhibitors (such as diisopropyl fluorophosphate, iodoacetic acid, etc.) can be added.S

The following section focuses on the selection of pH and salt concentration for the extraction solution.S

1. pH Value

Proteins and enzymes are amphoteric electrolytes with an isoelectric point. The pH of the extraction solution should be chosen within the pH range deviating from both sides of the isoelectric point. When extracting with dilute acid or dilute alkali, excessive acidity or alkalinity should be avoided to prevent changes in the dissociable groups of proteins, which could lead to irreversible changes in protein conformation. Generally, basic proteins are extracted with slightly acidic extraction solutions, while acidic proteins are extracted with slightly alkaline extraction solutions.S

2. Salt Concentration

Dilute concentrations can promote protein dissolution, known as salting-in. At the same time, dilute salt solutions, due to the partial binding of salt ions to proteins, have the advantage of protecting proteins from denaturation. Therefore, a small amount of neutral salt such as NaCl is added to the extraction solution, generally at a concentration of 0.15 mol/L. Buffer solutions often use 0.02-0.05M phosphate and carbonate isotonic salt solutions.S

(II) Organic Solvent Extraction Method

Some proteins and enzymes that are tightly bound to lipids or have more non-polar side chains in their molecules are insoluble in water, dilute salt solutions, dilute acids, or dilute alkalis. Organic solvents such as ethanol, acetone, and butanol can be used. These solvents have certain hydrophilicity and strong lipophilicity, making them ideal extraction solutions for lipoproteins. However, operation must be performed at low temperatures. Butanol extraction method is particularly superior for extracting some proteins and enzymes tightly bound to lipids, primarily because butanol has strong lipophilicity, especially its ability to dissolve phospholipids; secondly, butanol also has hydrophilicity, and within its solubility range (10% at 20 degrees Celsius, 6.6% at 40 degrees Celsius), it will not cause enzyme denaturation and inactivation. Additionally, the butanol extraction method has a wide range of pH and temperature choices and is suitable for animal, plant, and microbial materials.S

II. Protein Separation and Purification

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

(I) Separation Methods Based on Different Protein Solubility

1. Protein Salting Out

Neutral salts significantly affect protein solubility. Generally, at low salt concentrations, as salt concentration increases, protein solubility increases; this is called salting-in. When the salt concentration continues to rise, protein solubility decreases to varying degrees, and proteins precipitate sequentially; this phenomenon is called salting-out. When a large amount of salt is added to a protein solution, high concentrations of salt ions (such as SO4 and NH4 from ammonium sulfate) have a strong hydration force, which can strip the hydration layer from protein molecules, causing them to 'dehydrate.' Consequently, protein micelles coagulate and precipitate out. The effect of salting out is better if the solution pH is at the protein's isoelectric point. Because different proteins have different molecular sizes and degrees of hydrophilicity, the salt concentrations required for salting out also vary. Therefore, adjusting the neutral salt concentration in a mixed protein solution can cause various proteins to precipitate in fractions.S

Factors affecting salting out include: (1) Temperature: Except for temperature-sensitive proteins that operate at low temperatures (4°C), it can generally be carried out at room temperature. Generally, lower temperatures decrease protein solubility. However, some proteins (such as hemoglobin, myoglobin, albumin) have lower solubility at higher temperatures (25°C) than at 0°C, making them easier to salt out. (2) pH value: Most proteins have the lowest solubility in concentrated salt solutions at their isoelectric point. (3) Protein concentration: When protein concentration is high, the protein to be separated often co-precipitates with other proteins (co-precipitation phenomenon). Therefore, before salting out, serum should be diluted with an equal volume of physiological saline to make the protein content 2.5-3.0%.

Commonly used neutral salts for protein salting out mainly include ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride, sodium phosphate, etc. Ammonium sulfate is the most widely used among them, its advantages being a small temperature coefficient and high solubility (saturated solution at 25°C is 4.1M, i.e., 767 g/L; saturated solubility at 0°C is 3.9M, i.e., 676 g/L). Within this solubility range, many proteins and enzymes can be salted out. Additionally, ammonium sulfate's fractional salting out effect is better than that of other salts, and it is less likely to cause protein denaturation. The pH of ammonium sulfate solution is usually between 4.5-5.5; when salting out at other pH values, it needs to be adjusted with sulfuric acid or ammonia water.

After proteins are separated by salting out precipitation, the salt needs to be removed from the protein. A common method is dialysis, which involves placing the protein solution into a dialysis bag (commonly cellophane) and dialyzing it with a buffer solution, continuously changing the buffer. Since dialysis takes a long time, it is best performed at low temperatures. Additionally, desalting can be achieved by passing the solution through a column of dextran gel G-25 or G-50, which takes a relatively shorter time.

2. Isoelectric Point Precipitation Method

Proteins have the minimum electrostatic repulsion between particles at their isoelectric state, thus also having the minimum solubility. Since different proteins have different isoelectric points, one can adjust the solution's pH to reach a specific protein's isoelectric point to precipitate it. However, this method is rarely used alone and can be combined with the salting out method.

3. Low-Temperature Organic Solvent Precipitation Method

Using water-miscible organic solvents such as methanol, ethanol, or acetone can decrease the solubility of most proteins, causing them to precipitate. This method has higher resolving power than salting out, but proteins are more prone to denaturation, so it should be carried out at low temperatures.

(II) Separation Methods Based on Differences in Protein Molecular Size

1. Dialysis and Ultrafiltration

Dialysis separates proteins of different molecular sizes using a semi-permeable membrane.

Ultrafiltration uses high pressure or centrifugal force to force water and other small solute molecules through a semi-permeable membrane, while proteins are retained on the membrane. Filters with different pore sizes can be selected to retain proteins of different molecular weights.

2. Gel Filtration Method

Also known as size exclusion chromatography or molecular sieve chromatography, this is one of the most effective methods for separating protein mixtures based on molecular size. The most commonly used packing materials in columns are dextran gel (Sephadex gel) and agarose gel.

(III) Separation Based on Protein Charge Properties

Proteins exhibit different charge properties and charge quantities in different pH environments, allowing them to be separated.

1. Electrophoresis Method

Under the same pH conditions, various proteins can be separated due to their different molecular weights and charge quantities, leading to different migration rates in an electric field. Isoelectric focusing electrophoresis is particularly noteworthy; it uses an amphoteric electrolyte as a carrier. During electrophoresis, the amphoteric electrolyte forms a pH gradient that gradually increases from the positive to the negative electrode. When proteins with a certain charge migrate within this gradient, they stop upon reaching the pH position corresponding to their respective isoelectric points. This method can be used for analyzing and preparing various proteins.

2. Ion Exchange Chromatography Method

Ion exchangers include cation exchangers (e.g., carboxymethyl cellulose; CM-cellulose) and anion exchangers (diethylaminoethyl cellulose; DEAE-cellulose). When the protein solution to be separated flows through an ion exchange chromatography column, proteins carrying a charge opposite to that of the ion exchanger are adsorbed onto it. Subsequently, the adsorbed proteins are eluted by changing the pH or ionic strength. (See Chromatography Techniques chapter for details)

(IV) Separation Method Based on Ligand Specificity - Affinity Chromatography

Affinity chromatography is an extremely effective method for protein separation. It often requires only a single step to separate a target protein from a highly complex protein mixture, yielding high purity. This method is based on the specific, non-covalent binding of certain proteins to another molecule called a ligand. Its basic principle is that proteins exist as complex mixtures in tissues or cells, with each cell type containing thousands of different proteins. Therefore, protein separation (Separation), purification (Purification)

and characterization (Characterization) are important parts of biochemistry. To date, there is no single or ready-made method that can extract any type of protein from a complex mixture of proteins; therefore, several methods are often used in combination.

Cell Disruption

1. High-speed Tissue Homogenization: Prepare the material into a thin paste, place it in the cylinder to about 1/3 volume, close the lid tightly, set the speed regulator to the slowest position first, and then gradually accelerate to the desired speed. This method is suitable for animal visceral tissues, fleshy plant seeds, etc.

2. Glass Homogenizer Homogenization: First, place the chopped tissue into the tube, then insert the pestle and grind back and forth, moving it up and down to crush the cells. This method achieves a higher degree of cell disruption than a high-speed tissue homogenizer and is suitable for small quantities and animal organ tissues.

3. Ultrasonic treatment method: Use ultrasonic waves of a certain power to treat cell suspension, causing the cells to vibrate and rupture violently. This method is mostly suitable for microbial materials. For preparing various enzymes from E. coli, a concentration of 50-100 mg bacterial cells/ml is often chosen, and treated at a frequency of 1KG to 10KG for 10-15 minutes. The disadvantage of this method is that it generates a large amount of heat during the process, and corresponding cooling measures should be taken. Use with caution for ultrasonic-sensitive substances and nucleic acids.

4. Repeated freeze-thaw method: Freeze cells below -20 degrees Celsius, thaw at room temperature, and repeat several times. Due to the formation of ice crystals inside the cells and the increased salt concentration of the remaining cell fluid, swelling occurs, causing cell structure to rupture.

5. Chemical treatment method: For some animal cells, such as tumor cells, cell membranes can be disrupted using sodium dodecyl sulfate (SDS), sodium deoxycholate, etc. Bacterial cell walls are thicker, and treatment with lysozyme yields better results.

Regardless of the method used to break tissue cells, intracellular proteins or nucleic acid hydrolases will be released into the solution, leading to the biodegradation of macromolecules and a reduction in the quantity of natural substances. Adding diisopropyl fluorophosphate (DFP) can inhibit or slow down autolysis; adding iodoacetic acid can inhibit the activity of proteases whose active centers require sulfhydryl groups; adding phenylmethylsulfonyl fluoride (PMSF) can also eliminate proteolytic activity, but not completely. Additionally, conditions such as pH, temperature, or ionic strength can be selected to suit the extraction of the target substance.

Concentration, Drying, and Preservation

I. Sample Concentration

During the preparation of biomacromolecules, samples often become very dilute due to column purification. For the purpose of preservation and identification, concentration is often required. Common concentration methods include:

1. Vacuum heating evaporation concentration

By reducing the liquid surface pressure, the boiling point of the liquid is lowered. The higher the vacuum degree of the reduced pressure, the lower the boiling point of the liquid, and the faster the evaporation. This method is suitable for the concentration of some heat-sensitive biomacromolecules.

2. Airflow evaporation concentration: The flow of air can accelerate liquid evaporation. A thin layer of solution can be exposed to a continuous airflow on its surface; or, the biomacromolecule solution can be placed in a dialysis bag inside a cold room, and a fan can be used to blow air, allowing the solvent outside the membrane to evaporate without permeating, thereby achieving the purpose of concentration. This method has a slow concentration rate and is not suitable for concentrating large volumes of solution.

3. Freezing method: Biomacromolecules form ice at low temperatures, while salts and biomacromolecules do not enter the ice and remain in the liquid phase. During operation, first cool the solution to be concentrated to below its freezing point to solidify it, then slowly thaw it. The difference in melting points between the solvent and solute is utilized to remove most of the solvent. For example, when concentrating salt solutions of proteins and enzymes by this method, pure ice crystals free of proteins and enzymes float on the liquid surface, while proteins and enzymes are concentrated in the lower layer of the solution. By removing the upper ice chunks, a concentrated solution of proteins and enzymes can be obtained.

4. Absorption method: This method involves directly removing solvent molecules from the solution using an absorbent to achieve concentration. The absorbent used must not chemically react with the solution, not adsorb biomacromolecules, and be easily separable from the solution. Commonly used absorbents include polyethylene glycol, polyvinylpyrrolidone, sucrose, and gels. When using polyethylene glycol as an absorbent, first place the biomacromolecule solution into a semi-permeable membrane bag, cover it externally with polyethylene glycol, and place it at 4 degrees Celsius. The solvent seeping out of the bag is quickly absorbed by the polyethylene glycol. After the polyethylene glycol becomes saturated with water, it should be replaced with fresh absorbent until the desired volume is reached.

5. Ultrafiltration method: Ultrafiltration is a method that uses a special membrane to selectively filter various solute molecules in a solution. When a liquid passes through the membrane under a certain pressure (nitrogen pressure or vacuum pump pressure), the solvent and small molecules pass through, while large molecules are retained. This is a new method developed in recent years, most suitable for the concentration or desalting of biomacromolecules, especially proteins and enzymes. It has advantages such as low cost, convenient operation, mild conditions, better preservation of biomacromolecule activity, and high recovery rates. The key to applying ultrafiltration is membrane selection. Different types and specifications of membranes have different parameters such as water flow rate and molecular weight cut-off (i.e., the approximate minimum molecular weight value that can be retained by the membrane), and must be selected according to work requirements. In addition, the form of the ultrafiltration device, the composition and properties of the solute, and the solution concentration all have a certain impact on the ultrafiltration effect. Molecular weight cut-off values for Diaflo ultrafiltration membranes:

Membrane Name; Molecular Weight Cut-off; Average Pore Diameter

XM-300; 300,000; 140

XM-200; 100,000; 55

XM-50; 50,000; 30

PM-30; 30,000; 22

UM-20; 20,000; 18

PM-10; 10,000; 15

UM-2; 1,000; 12

UM0550010

Hollow fiber tubes are made from the above ultrafiltration membranes, and many such tubes are bundled together. Both ends of the tubes are connected to a low ionic strength buffer solution, allowing the buffer to flow continuously within the tubes. The fiber tubes are then immersed in the protein solution to be dialyzed. When the buffer flows through the fiber tubes, small molecules easily pass through the membrane and diffuse, while large molecules cannot. This is the fiber filtration dialysis method. Due to the increased dialysis area, the dialysis time is shortened by 10 times.

II. Drying

After preparation, biomacromolecule products often require drying to prevent deterioration and facilitate preservation. The most commonly used methods are freeze-drying and vacuum drying. Vacuum drying is suitable for drying and preserving substances that are heat-sensitive and easily oxidized. The entire setup includes a dryer, condenser, and the principle of vacuum drying, with the added factor of temperature. Under the same pressure, water vapor pressure decreases with decreasing temperature; thus, at low temperature and low pressure, ice easily sublimates into gas. During operation, the liquid to be dried is generally first frozen to below its freezing point to solidify it, and then the solvent is removed by turning it into gas under low temperature and low pressure. Products dried by this method have advantages such as being loose, having good solubility, and retaining their natural structure, making them suitable for the dry preservation of various biomacromolecules.

III. Storage

The stability of biological macromolecules is greatly related to their preservation methods. Dry products are generally more stable, and their activity may not change significantly for several days or even years at low temperatures. Storage requirements are simple: just seal the dry sample in a desiccator (containing a desiccant) and keep it in a 0-4 degree refrigerator. When storing in liquid form, the following points should be noted:

1. The sample should not be too dilute; it must be concentrated to a certain concentration before encapsulation and storage. Samples that are too dilute can easily denature biological macromolecules.

2. Generally, preservatives and stabilizers need to be added. Commonly used preservatives include toluene, benzoic acid, chloroform, thymol, etc. Commonly used stabilizers for proteins and enzymes include ammonium sulfate paste, sucrose, glycerol, etc. For enzymes, substrates and coenzymes can also be added to improve their stability. In addition, solutions of calcium, zinc, boric acid, etc., also have a certain protective effect on some enzymes. Nucleic acid macromolecules are generally preserved in standard buffer solutions of sodium chloride or sodium citrate.

3. Low storage temperature is required, mostly stored in a refrigerator around 0 degrees, and some require even lower temperatures, depending on the substance.