Analysis of Wheat Peroxidase Isoenzymes by Vertical Polyacrylamide Gel Electrophoresis

In biology, isozymes can be used to study species evolution, genetic variation, cross-breeding, ontogeny, tissue differentiation, etc. Genetic variation in animals and plants can be identified by comparing the isozyme profiles of offspring and parents.

Peroxidase is a highly active enzyme commonly found in plants. It is related to respiration, photosynthesis and auxin oxidation. Its activity changes continuously during plant growth and development. Therefore, measuring the activity of this enzyme or the changes in its isoenzymes can reflect the changes in plant metabolism in a certain period.

Principle

Principle of discontinuous polyacrylamide gel electrophoresis

The discontinuity of the system is manifested in the following aspects:

• The gel plate consists of upper and lower layers of gel, and the pore sizes of the two layers of gel are different. The upper layer is a large-pore stacking gel, and the lower layer is a small-pore separation gel.
• The ionic composition of the buffer and the pH of each gel layer are different. This experiment uses an alkaline system. The electrode buffer is Tris-glycine buffer with pH 8.3, the stacking gel is Tris-HCl buffer with pH 6.7, and the separating gel is Tris-HCI buffer with pH 8.9.
• A discontinuous potential gradient is formed in the electric field. In such a discontinuous system, there are three physical effects, namely charge effect, molecular sieve effect and concentration effect. Under the combined action of these three effects, the substances to be tested are well separated.

Taking the wheat seedling peroxidase isoenzyme to be isolated in this experiment as an example, the three effects are explained respectively.

• Charge effect: Various enzyme proteins swim toward a certain electrode at a certain speed under the action of an electric field according to the type and amount of charge they carry.
• Molecular sieve effect: Molecules with small relative molecular mass and spherical shape experience less resistance during electrophoresis and move faster. On the contrary, molecules with large relative molecular mass and irregular shapes will experience greater resistance during electrophoresis and move slower. This effect is different from that seen during gel filtration.
• Concentration effect: Each component in the sample to be separated will be compressed into layers in the stacking gel, making the originally dilute sample highly concentrated. The reasons are as follows:
  Due to the different pore sizes of the two gels, when the enzyme protein moves downward to the interface of the two gels, the resistance suddenly increases and the speed slows down. The band of the enzyme protein to be separated at the interface becomes narrower and the concentration increases.
  In polyacrylamide gel, although the stacking gel and the separation gel use Tris-HCl buffer, the pH of the upper stacking gel is 6.7 and the pH of the lower separation gel is 8.9. HCl is a strong electrolyte. No matter which layer of gel it is in, HCl is almost completely ionized, and CI covers the entire gel plate. The enzyme protein sample to be separated is added to the sample tank and immersed in pH 8.3 and Tris-glycine buffer. At the beginning of electrophoresis, CI with the largest effective migration rate quickly runs to the front and becomes a fast ion. Under the condition of pH 6.7, glycine (pl=6.0) with a dissociation degree of only 0.1% to 1% has the lowest effective swimming rate and runs at the end, becoming a slow ion. In this way, a constantly moving interface is formed between fast ions and slow ions. Under the condition of pH 6.7, the enzyme protein with negative charge has an effective mobility rate between fast and slow ions, and is clamped and distributed near the interface, gradually forming a zone. Since fast ions move forward rapidly, the part of the area where they originally stayed becomes a low ion concentration area, that is, a low conductivity area. Because the potential gradient V, the current intensity I and the conductivity S have the following relationship: V=I/S Therefore, under constant current conditions, a higher potential gradient is generated on both sides of the low conductivity area. This high potential gradient generated after the start of electrophoresis acts on enzyme proteins and glycine slow ions to accelerate forward and catch up with fast ions. The enzyme protein band originally sandwiched between fast and slow ions is gradually compressed and aggregated into a narrower band during this pursuit. This is the so-called concentration effect. In this zone, various enzyme proteins are divided into different levels according to their charges, and are initially separated before entering the separation gel, forming several "starting lines" that are very close but different.
  When both enzyme proteins and slow ions enter the separation gel, the pH changes from 6.7 to 8.9, the dissociation degree of glycine increases sharply, and the effective mobility increases rapidly, thus catching up with and surpassing all enzyme protein molecules. At this time, the interface between fast and slow ions runs in front of the separated enzyme protein, and the discontinuous high potential gradient no longer exists. Therefore, in the subsequent electrophoresis process, the enzyme protein is separated only by the charge effect and the molecular sieve effect under a uniform potential gradient and pH conditions. Compared with the continuous system, the resolution of the discontinuous system is greatly improved, so it has become a widely used separation analysis method.

Principle of identification of peroxidase isoenzymes

Isoenzymes are proteins from different tissues of the same organism or different subcellular structures of the same cell that can catalyze the same reaction. Isoenzymes are the products of gene encoding. Due to the template function, the amino acid sequence on the polypeptide chain in the enzyme protein directly reflects the sequence of base pairs on the DNA chain. The changes can represent changes at the DNA molecular level, so isoenzyme analysis can be interpreted as an effective means to study the genetic differentiation of biological populations from the protein molecular level. The expression of enzymes in organisms is directly controlled by genetic genes. The enzymes are separated by the concentration of polyacrylamide gel, the molecular sieve effect and the charge effect of electrophoretic separation. After electrophoresis of the crude enzyme solution extracted from wheat seedlings, specific enzyme staining was performed. Different enzyme components were displayed in different positions of the gel, presenting a biologically unique isoenzyme spectrum.

The principle of selecting wheat as sample

Isoenzymes are the direct products of genes and have obvious species, tissue and developmental stage specificity. POD isoenzyme is a common oxidase in plants. It coordinates with superoxide dismutase (SOD) and catalase to remove excess free radicals and maintain free radicals in plants at a dynamic and normal level to improve plant stress resistance.

In the later stages of wheat growth and development, the plants tend to age, the grains mature, and the content of free radicals and their derivatives in the body continues to increase. In particular, the increase in hydrogen peroxide, the product of SOD oxidation free radicals, requires a large amount of POD to decompose hydrogen peroxide. The increase in POD activity in the later stages of biological development may also be used to decompose chlorophyll and auxin to stop plant growth as early as possible and reduce nutrient consumption, thereby strengthening the plant's resistance to damage caused by free radicals and their derivatives and the adverse natural environment.

Procedures

1. Installation of electrophoresis tank

Place the two glass plates correctly into the silica gel strips, clamp them in the electrophoresis tank, and tighten the screws in diagonal order, paying attention to balanced force to avoid breaking the glass plates. Install the electrophoresis tank and seal the bottom with 2% agar solution prepared with pH 8.9 buffer. After the agar solidifies, the gel can be cast.

2. Preparation of gel

After mixing in a small beaker, pour the gel immediately. When filling the gel, tilt the electrophoresis tank slightly, place the tip of the small beaker against a certain point in the center of the top of the long glass piece, and carefully pour it between the two glass pieces until it is about 2 cm away from the top of the short glass piece. Level the electrophoresis tank, and immediately use a dropper to carefully and gently cover the upper layer of the gel with a 2-3 mm thick water layer. When pouring the water layer, pour it evenly and gently to prevent holes from forming on the top of the gel and affecting the result. When water is first added, the interface can be seen, and then gradually disappears. When the interface reappears, it indicates that the separation gel has polymerized. Let it sit for a while, then pour out the water, use a filter paper strip to soak it slightly from one side, be careful not to damage the gel surface, and then prepare to pour the concentrated gel.

After the preparation is uniform, pour the gel immediately, and the operation is the same as pouring the separation gel. When the gel surface reaches about 0.5 cm from the short glass piece, insert the sample slot template (comb) immediately. After the polymerization is completed, pour the electrode buffer into the electrophoresis tank, then carefully pull out the comb and prepare for spotting.

3. Spot samples

Use a microsampler to carefully draw the sample solution into the sample tank.

Sample loading volume: Both leaf sample liquid and root sample liquid were loaded in gradient, which were 5 µL, 15 µL, 30 µL, and 50 µL respectively.

4. Electrophoresis

Connect the power cord (the upper slot is connected to the negative pole, and the lower slot is connected to the positive pole). Turn on the power switch and adjust the current to about 15 mA initially. When the leading edge enters the separation gel, the current can be adjusted to about 30 mA. Stop the electrophoresis when the leading edge indicator dye moves down to 1-2 cm from the end of the gel plate.

5. Stripping, dyeing and recording results

After the vertical polyacrylamide gel electrophoresis is completed, remove the vertical plate from the electrophoresis tank, carefully separate the two glass plates, and carefully place the gel into a large petri dish for staining reaction. Peroxidase generates free oxygen radicals during the decomposition of hydrogen peroxide, which can cause a color reaction in Anise and produce a brown compound. Therefore, on a gel plate that has been soaked in dihydranisidine staining solution, brown bands can be seen where there are peroxidase isoenzyme protein bands.

The dyeing process is as follows:

A. Pour about 50 mL of pH 4.7 acetic acid buffer into a large petri dish, submerge the gel plate, and soak at room temperature for 6-7 min.

B. Use a funnel to recover the acetic acid buffer, add staining solution, submerge the gel plate, and soak at room temperature for 20 min. During this period, bands of peroxidase isoenzymes will appear, and the enzyme spectrum will be observed and recorded.

Note

• Acrylamide and methylene bisacrylamide are nerve agents that can be absorbed through the skin, and their effects are cumulative. When weighing powdered acrylamide and methylene bisacrylamide, gloves and masks should be worn, and purification should be carried out in a fume hood.
• Do not touch the glass on the glued surface with your hands to prevent the gel plate and glass plate from peeling off, causing bubbles and slippery gel, or the gel plate breaking when peeling off the gel. All equipment used should be strictly cleaned.
• There should be no air bubbles when sealing with agarose and filling the gel, so as not to affect the flow of current during electrophoresis, thus affecting the shape and migration direction of the DNA band.
• After removing the spotting comb from the gel, clean the sampling well immediately and thoroughly, otherwise the small amount of acrylamide solution retained by the comb will polymerize in the sampling well, resulting in an irregular surface and deformation of the DNA band.
• After the gel is completely solidified, it must be left for about 30 min to allow it to fully age before the sample comb can be gently removed. Do not damage the flatness of the bottom of the sample well to avoid distortion of the zone after electrophoresis.

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