A method of obtaining the enzyme peroxidase from horseradish roots. Method of obtaining peroxidase from horseradish roots Electrochemical behavior of horseradish peroxidase substrates

"Hacked" dissertation

There was no need to search for the topic. All his childhood was associated with horseradish. The village grandmothers had no pills. Health was improved by the gifts of nature. And as soon as the snow melted, they went to the garden to dig a healing root. He wrote and hacked his dissertation himself.

I thought that I licked the sour cream without revealing the essence. I missed something important for agricultural technology, - the scientist explains his extraordinary act.

In pursuit of the truth, he reasoned something like this. In the homeland of horseradish, in Russia, for 60 years it was practically written off. Of the 500 existing varieties, only 2-3 remain. And in Europe, where it appeared 200 years ago, today there are 2,500 varieties. In America, where it was brought in 1900 at the initiative of the Ministry of Agriculture, there are already 3.5 thousand. And work on breeding it continues. What for?

The first half-closed information that horseradish can be used to cure cancer was leaked from Israel. Later, America and Europe appeared. I turned to scientists from the FMD Institute and the Institute of Microbiology. They deciphered the formula of the drug - peroxidase. Only half a gram was obtained from a ton of mashed horseradish. He began to pester him, how to "touch" him and see, - says Emelin.

It was just right to involve intelligence officers in the case. And so it happened. With their help, we went to a French company that traded our oil. Clicked. They promised to build a peroxidase plant. But the technology for its production remained under lock and key.

Right from the field, so that the most valuable thing did not have time to evaporate, the state farm horseradish was sent for analysis to a laboratory in France. The analysis is lost. They sent it a second time.

And it turned out that in our horseradish the peroxidase content is 50 times higher than in imported one. That is, from one ton we can get not 0.5 grams, but 25 grams. "You will bring out at least 30 thousand varieties of horseradish, but you will never get one like we have in Russia. This is our property," Emelin triumphed then.

It turned out that the peroxidase content in our horseradish is 50 times higher than in imported one.

And the defense of his dissertation on horseradish still took place. The scientific supervisor was Marina Vladimirovna Alekseeva, a student of Ivan Vladimirovich Michurin, a professor at the Agricultural Institute in Balashikha. Opponents - luminaries of vegetable growing, who could hardly believe that some collective farmer from the Vladimir provinces swung at such a topic. The result of the defense was a methodological manual for the industrial cultivation of horseradish. Meanwhile, scientists from two Vladimir institutes, FMD and Virology, to which Emelin supplied raw materials from his farm, created a technology for producing domestic peroxidase.

We were on the verge of industrial production of a substance, the price of which on the world market reached 7 thousand dollars per gram. If 100 years ago the people of Suzdal traded with Germany very profitably, sending there 40 wagons of horseradish roots a year, then this technology could become a gold mine for the country, - Yuri Anatolyevich is convinced.

But perestroika began, and the country had no time for shit. Scientific research was curtailed, and the scale of its industrial cultivation was reduced to one field in the "Stavrovsky" state farm, whose director by that time was Yemelin.

Garden Serpent Gorynych

Or maybe it's good that we limited ourselves to one field? - I think out loud, remembering how difficult it is to fight him. It is worth getting horseradish in the garden, you will never get it out. The story of the famous artist, and now the governor, Mikhail Evdokimov, comes to mind about how he tried to get rid of the hell, but even the tol did not help.

What then is to be done?

Don't fight him. It can only be tamed. Horseradish gets along well with onions, potatoes and other garden crops and protects them from pests. Our ancestors noticed this long ago. Even 500 years ago, they said: a garden without horseradish is like a herd without a shepherd. And when they started gardens, first of all they determined the place where horseradish would grow.

It is no coincidence that horseradish and onion plantations were on the Suzdal land. First they removed the onions, and then the horseradish. It's the same with potatoes. In September it was dug up, and in October the horseradish came up. We sowed wheat and rye for horseradish. And they received 50-60 centners per hectare, - says Emelin.

Studying horseradish, Emelin came to "archaeological" excavations. In the area where he grew up, he sifted the ground and counted the roots. And he concluded that there are two types of horseradish. Rooted to a depth of 15 meters, he called maternal. Formed in the upper layer of soil from small roots left after harvesting - upstream.

The real horseradish is maternal. He takes out from the subsoil layers, the so-called Adam's earth, those nutrients and elements that are no longer here. But horseradish weed can also become maternal. Give me time, because in a year it goes into the ground by 60-70 cm.

So, "infection" with horseradish is quite likely? - I express my concern to Emelin. And he responds with a new argument:

Horseradish is a self-regulating culture. Just as you cannot pour two barrels of water into one empty barrel, so on one piece of land it will not grow more than it should.

How much is it?

Its yield is a maximum of 4-5 tons of roots per hectare. Such a sharp limitation of the scope of productivity distinguishes horseradish from other crops. I say this with full responsibility as a person who has devoted more than 30 years to studying it. But if he settled, then for centuries. On the land where horseradish is grown, he will be born for 100 or even 300 years.

So, Mikhail Evdokimov is right and only a nuclear bomb will save him from him? - I continue the argument on behalf of all the gardeners.

You won't cut the apple tree after you have picked the apples, will you? She will harvest next year. So it is with horseradish. Previously, it was dug in the month whose name contains the letter "p": September, October, November (the rest of the time it does not have that pungency and mustard smell) ... Cut off the top as much as you can, and by the next season on this place will grow even more.

When in Russia they talked about the Serpent Gorynych, in which three grow back in place of one cut off head, they probably meant horseradish, - explains Emelin.

Behold at the root

SPK "Stavrovsky" is one of the rare places in Russia where you can see a field of blooming horseradish. And they say that bees fly over him for days. True, the director did not have to eat shitty honey. But he gave flowers. They, Emelin believes, are extraordinary beauty.

And horseradish seeds are formed. But if they are sown, nothing will come up. Horseradish propagates only with the help of roots - vegetatively. Why? Emelin did not find an answer. But on the other hand, this is one of the moments that allows a scientist to think about its ancient origin. Having survived the strongest cataclysms, the plant, apparently, thus adapted and survived.

There is still no exact answer: is it a vegetable, a medicinal plant or a spice? The lack of clear species boundaries is also, according to Emelin, confirmation of the hypothesis that horseradish is the messenger of Atlantis.

The origin of many cultures has long been known. Where the hell came from, there is a lot of confusion in science. In many sources I read that under Tutankhamun the Egyptians somewhere took the root worth its weight in gold from the barbarians. But I do not have a firm conviction that this is hell, - Yemelin does not hide.

But, deifying the subject of his research, he remains a practitioner. Having traveled about 250 vegetable gardens of the Vladimir and Yaroslavl regions in search of breeding material, he bred a new horseradish variety - "Tolpukhovsky" (after the name of the central estate of his farm) and planted its plantation on the steep bank of the Koloksha River. Says: until the next civilization.

Emelin himself uses horseradish as a medicine. But unlike us, uneducated ones, he takes into account that the beneficial properties in mashed horseradish persist for only 7 days.

In the horseradish, which for months "dangles" on store shelves, and in our closets, there is not a damn thing! - says the scientist.

What to do?

They dug 5-6 kilograms out of the ground in the fall, put them in the cellar, sprinkled them with earth, and let them lie there. Take out a little from there to make a 250 gram jar. It can be taken twice a year: in winter and early spring. In winter - from the cellar. And in the spring, a little the earth thawed, you can safely dig up until the leaves grow up to 5 centimeters. This is what our ancestors did when they ate a piglet with fresh horseradish on Easter.

Emelin can be trusted. He recently released three books one after another: "Horseradish in your garden", "Horseradish your doctor" and "Horseradish on your table". Now he is preparing for publication "Khrenolechebnik". And he continues to refute the stereotype that shitty means bad.

Or maybe, really, when they say that life is absolutely bad, they mean only the fate of the Russian horseradish?

"There are a number of cancer sites that are practically not cured. For example, when
metastases spread to the liver, and in such cases even chemotherapy does not help. And only in rare cases, metastases can be removed surgically. But we knew that horseradish peroxidase increases the body's immunity, and this is the main condition for treatment. This very peroxidase has even been learned to obtain in its pure form. But it turned out to be a crazy idea - 5 grams came out of a ton of raw materials! I once read in the three-volume Immunology, published in the USSR back in 1974, that the ingestion of horseradish peroxidase into the blood increases the effectiveness of treatment 4 thousand times! But only after many years did he figure out how to introduce it - through an enema!
Here's how it's done
Grate horseradish on the finest grater. Take 1 tbsp. a spoonful of horseradish and pour half a glass of boiled water. Leave in the refrigerator for 12 hours. Take a baby enema and after stool enter 30-40 grams. But first, do a cleansing enema.
Thus, the tincture is absorbed instantly and immediately enters the liver. On the surface of leukocytes in cancer patients there are receptors that perceive horseradish peroxidase, and then the activation of killer leukocytes increases four thousand times.
After 10-15 procedures on the control scan, cancer metastases were not fixed or stopped. Do an enema with horseradish every other day.
It is more difficult to treat lung cancer with horseradish peroxidase. The best way is inhalation, but it will be effective if you grind horseradish to 3-5 microns, everything that is larger is not absorbed by the body.
The Institute of Biological Instrumentation has developed special spinhiller inhalers that can grind horseradish and prepare hundreds of inhalations. Developed, but not produced, because in this case, horseradish becomes a drug! And this is seven years of testing, years of affirmations, etc.
But the pharmaceutical committee does not prohibit breathing, rub and breathe, already in the very process of rubbing you will breathe enough, and then, covering yourself with a towel, breathe over the manufactured medicinal product. The procedure will take 2-3 minutes. And maybe I can wait for spinhillers - I’m only 72!

So, you rub the roots of horseradish and breathe for 1-2 minutes until tears flow from your eyes. This should be done daily, until complete recovery.
Once a reader who suffered from chronic pneumonia called us at the editorial office. She is 58 years old, she was cured in this way in less than a month.

"- Leonid Nikolayevich J. from Germany, he has metastases in the liver. And this organ is practically not treated, however experiments with microclysters from horseradish showed positive results. Leonid Nikolayevich tried it, but felt a strong burning sensation and could not hold the liquid for a long time. can I advise?
- Obviously, the person did too much concentration. Despite the fact that the enema should not exceed 30 grams, it does not need to be held, and the liquid itself will remain there. Before the procedure, if you did not have a stool, you should do a cleansing enema. In total, you should spend 10-15 sessions every other day, and upon completion, see a doctor ...

Here is a call from Germany, from Essen. Leonid Zhuravsky (I already mentioned him when we wrote about enemas with horseradish infusion, he had some problems with their use) is seriously ill, the liver is already affected by metastases. It is clear that in such cases you speak with extreme caution, because you can expect anything. And suddenly I hear: “The growth of metastases has stopped, will you speak or see Dr. Laskin, convey my sincere gratitude to him. Thank you for your publications. "

X
.... “I returned from vacation and found out that Nina was denied treatment. I found the FiS magazines with articles by Dr. Laskin, and we began to use his methods of treatment with horseradish tinctures and buckwheat. The problem was that Nina practically did not eat anything and could not even drink. She suffered from constant nausea and vomiting, even after taking water. The first horseradish enema caused a burning sensation, and the subsequent ones were painless, a total of 10 procedures were already done. Nina began to eat a little buckwheat porridge with a little olive oil. She drank two raw eggs yesterday. The body accepted the first egg (it was at lunchtime) normally, and Nina said: “I felt so good!” ....
I am asking you for help. We would like to get advice from Dr. Laskin and other specialists, and as soon as possible, since the bill does not go on for months, every day is expensive.
Is it possible to do enemas with horseradish more than 15 times?
.... We contacted the co-author of the diet, Alexander Balura. By the way, he is now closely engaged in simplifying the use of the healing properties of horseradish, or rather, the peroxidase contained in it, it activates macrophages that destroy cancer cells. So, Baliura is experimenting with imprisoning horseradish in a sealed test tube, where both the liquid and the spirit will be preserved, so to speak. Indeed, in such megalopolises as Moscow, for example, finding a horseradish root is a problem, if there is no site outside the city, and if there is, it rarely grows with anyone, and the city authorities have long eliminated the grannies who traded near the metro.
Here is what A. Baliura says: “In such a difficult situation as with a friend of the author of the letter, you can continue to do enemas from horseradish tincture and further. You can reduce the amount of liquid to 5 ml (this is just a tablespoon of the solution). On condition of competent preparation: fine grater, dilution with cold boiled water, settling in the refrigerator for 10-12 hours. "
X

But a woman from the Donetsk region is worried about her father. They live in different cities of Ukraine, and the daughter gives advice to her father and his family, apparently by phone. My father's stomach and liver are affected, but after eating buckwheat and microclysters from infused horseradish, the liver shrank to its normal size. But it turned out that the process also moved to the lung, and now he regularly breathes in vapors of grated horseradish.
X

Several questions of a "technological nature". Reader M. from Tula asks why horseradish for administration through an enema must be insisted and kept in the refrigerator for 12 hours? Is there a minimum and a maximum? Explain: 10-12 hours is a laboratory proven time. You need to grate only 1 tbsp. a spoonful of horseradish, but do not do it for future use, every time it must be freshly made.
One of the readers complains that after 12 hours the tincture becomes too cold, and asks if it is possible to dilute it with warm water? We answer: no, what kind of concentration will it be? Just keep the tincture at room temperature after the refrigerator for a while, and it will become acceptable for use.
More about horseradish. So far, it is known that it has a good effect on the liver, even when it is metastatic. In other cases, Laskin does not recommend using enemas.
But it is possible and necessary to breathe rubbed horseradish in case of illness not only of the lungs, but also of the bronchi, throat, nose, that is, the entire area of \u200b\u200bthe nasopharynx. To do this, it is enough to carry out the procedure twice a day for 2-3 minutes, until tears flow from the eyes. Let me remind you that in this way you can eliminate less severe ailments - flu, colds, runny nose. "

Answering the first letter, I can say the following. Horseradish enemas are, as practice has shown, the most effective way for healing substances to penetrate the liver to cope with metastases. Enemas should be done as follows: 1 tbsp. pour a spoonful of grated horseradish with 0.5 cups of chilled boiled water, and put all this in the refrigerator for 8-12 hours (but not in the freezer). Then strain the infusion and draw approximately 20 ml into an enema (small, for children) for introduction into the body. Horseradish gruel should never be injected into the rectum, as it is too concentrated, and this can lead to burns of the mucous membrane.
The rest of the strained infusion can be stored for another day in the refrigerator in order to make another enema from it the next day. Such enemas should be administered after bowel movement. You can do enemas daily, but every other day prepare a new infusion of horseradish in order to take full advantage of its anti-cancer properties.
I hope my answers will help the letter writers cope with the disease. We are very concerned about the results of the treatment. In any case, write about them in redaction.
With wishes of recovery,
Alexander BALURA, Candidate of Medical Sciences

Boil 1 kg of beets per 5 liters of water for 5-6 hours over low heat, you get a thick broth, dissolves stones w / n method, I checked my luck.

In # 8 of AiF-Health, 2008, I read that according to the Canadian Medical Association and the International Agency for Research on Cancer, many plant foods contain powerful anti-cancer agents. These are cabbage (cabbage, cauliflower, broccoli, Brussels sprouts), garlic, onions (onions and shallots), spinach, watercress, flax seeds, linseed oil, tomatoes, black pepper, turmeric, blueberries, raspberries, blackberries, blueberries, cranberries, grapes, dark chocolate, citrus fruits, green tea, red wine). Taking these foods daily can protect against cancer even in those with poor heredity. In this regard, I have two questions. If everything is so simple, why don't people in all countries eat these foods every day to protect themselves from cancer? Why doesn't this list include buckwheat and olive oil, which are the basis of Dr. Laskin's anti-cancer diet?
Marina L., Moscow
Not only buckwheat
The dietary method of cancer prevention and therapy has developed rapidly in recent years. A number of specialists working in this area even consider cancer to be a "disease of insufficiency", that is, a disease arising from insufficient consumption of special phytochemicals that are part of green tea, red wine, brown rice, berries, fruits, vegetables, mushrooms, nuts, seeds, spices. For thousands of years, the human body has adapted to this particular diet, and the transition in the 20th century to the diet of refined carbohydrates (white bread, white sugar, sweets, confectionery) tragically affected its health. Cancer has become one of the main diseases of civilization. Returning to a diet based on natural products allows the human body to effectively resist cancer.
A number of anti-cancer diets have been developed recently. In Western countries, special attention is paid to cranberries when it comes to food, and Mediterranean diets (vegetables, fruits, whole grain products, sea fish, olive oil, dry red wine).
So, recently in the United States, a study was conducted on the effect of the Mediterranean diet on the ability to inhibit the development of cancer in 23 patients aged 43-74 who refused radical treatment, with limited prostate cancer confirmed on a biopsy. On average, after 38.5 months of observation, 87% of men noted a 58% decrease in PSA (prostate specific antigen), which was a marker of cancer activity. And the 3 men in this study had a very small increase in PSA. These results indicate the significant effectiveness of the Mediterranean diet in reducing the progression of prostate cancer.
So, the combination of a number of natural products in the diet allows the body to control, prevent and even stop the cancer process. Why do people regularly eat not these amazingly healthy foods, but prefer unhealthy sweets, pastries, etc.? Yes, because they are much tastier and, moreover, like alcohol, cause addiction to themselves. This is a real food addiction, and it is akin to drug addiction. When there is still cancer, and different yummy right now will improve your mood, relieve stress and depression, and restore peace of mind. Try it, give it up! Until the thunder breaks out, the man does not cross himself. This is how we live, we are sick, and we die much earlier than the due date.
Now regarding the second question: why buckwheat porridge and olive oil are not included in the list of anti-cancer products? The fact is that buckwheat porridge is widely used mainly in Russia, Ukraine, Belarus. In the USA, Canada, Western Europe, it is eaten mainly by emigrants, and buckwheat is grown there not for food, but in order to extract vitamins, minerals and various vitamin-like substances from it, including quercetin.
Readers of "FiS" know that the anticancer properties of buckwheat porridge were first applied by our compatriot doctor-oncologist V.A. Laskin. It was over 35 years ago. Moreover, buckwheat porridge began to show its effect especially powerfully as part of a strict diet (buckwheat porridge, olive oil, rose hips, water). The results of its application, obtained by Laskin, left no doubt about it. Wolfe Laskin's wife, also a doctor by profession, recalled: “Returning home in the evening after a reception at which he was previously a cancer patient and now fully recovered (after all, many brought the conclusion of an oncological institution that they no longer had cancer), my husband could not to calm down the whole evening and until late at night sat over books, trying to understand why the buckwheat diet works so powerfully. " Realization came only in 2000, when American scientists determined that buckwheat contains 8% quercetin, one of the most powerful natural substances for the prevention and treatment of cancer.
As for olive oil, in my opinion, Dr. Laskin somewhat underestimated the role of olive oil in his diet. When I asked him why he chose olive oil, he replied that the inhabitants of the Mediterranean basin eat quite a lot of olive oil and have little cancer. This thought fascinated him, but since he did not know which components of olive oil are responsible for the anti-cancer effects, he, according to the logic of the Soviet doctor, thought more about buckwheat porridge with a record quercetin content.
Meanwhile, in recent years, it is in olive oil that unique antioxidants of phenolic and non-phenolic nature have been discovered, which are no less important than quercetin for fighting cancer. And now I am sure that the effectiveness of the Laskin diet is associated precisely with the combination of buckwheat porridge and a large amount of olive oil (6-8 tablespoons per day).
Now about the useful anti-cancer herbal products, the list of which is published in "AiF-Health". Almost all of them are part of Dr. Laskin's "weekly diet". That is, first, for 5-6 weeks (sometimes longer), the patient follows a strict diet, and then switches to a weekly diet, which, in addition to buckwheat porridge, olive oil and rose hips, includes blueberries, broccoli, shitaki mushrooms ... From the list of products listed in "AiF-Health", I would also add cranberries, turmeric and flaxseed to Laskin's menu.
Zinoviy BELKIN, Candidate of Medical Sciences

The method involves crushing and homogenizing the germinated horseradish roots, extracting the homogenized horseradish roots with 0.15 ± 0.01 M sodium chloride solution. Ballast proteins are separated and peroxidase is precipitated with ammonium sulfate salts of 45-48% saturation and 85-90% saturation, respectively. Gelfiltration of the peroxidase solution is carried out on Sephadex G-100 with elution with 0.15-0.2 M sodium chloride solution. Chromatographic purification is carried out on carboxymethyl cellulose. Freeze drying of the target peroxidase is carried out. Dialysis against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0 ± 0.1 are performed. Concentration is carried out with additional purification on DEAE-cellulose with elution with the same buffer, followed by dialysis against deionized water. EFFECT: method allows obtaining peroxidase with high specific activity and high yield. The peroxidase yield is 2.52-3.50 g / kg of horseradish roots, the specific activity is 640-700 EA / mg protein. 5 p.p. f-crystals, 2 dwg., 1 tab.

Drawings for RF patent 2353652

The invention relates to the field of biotechnology, in particular to the production of enzymes from plant materials, and can be used for laboratory and industrial production of the enzyme peroxidase from horseradish roots for immunology and immunochemistry as the main component of conjugates for enzyme immunoassays.

There are various methods for obtaining peroxidase from horseradish roots.

A known method of producing peroxidase from horseradish roots, including homogenization of horseradish roots, extraction of the enzyme with saline solution, precipitation of the enzyme with ammonium sulfate salts, gel filtration, alcohol precipitation, electrophoresis, reprecipitation with ammonium chloride, filtration through Sephadex G-50 and DEAE-cellulose.

The main disadvantages of this method are the low activity and purity of the resulting product, as well as the complexity of its preparation and a large number of stages of the technological process.

There is also known a method of obtaining peroxidase from horseradish roots, according to which horseradish roots are homogenized, then the enzyme is extracted with water and peroxidase is precipitated with ammonium sulfate salts, followed by gel filtration [US Pat. Hungary 172872, C07G 7/022].

The disadvantage of this method is the low yield and low purity of the enzyme.

The known method [A.S. Bulgaria 46675, C12N 9/08, 15.02.90], according to which horseradish roots are germinated for 2-3 days, then homogenized and the enzyme is extracted with water during the day. The aqueous extract is centrifuged, followed by fractionation of proteins with ammonium sulfate salts, then the ammonium sulfate precipitate of the enzyme is dissolved in distilled water and ultrafiltered on filters "Milipor" PTGC 000 05. 0.5 M phosphate buffer (pH 8) is added to the resulting filtrate in a ratio of 100 parts of buffer per 1 part of the filtrate and passed through a column with DEAE-Sephadex A-50 ion exchanger, then successively ultrafiltered on Milipor PTGC 000 05, RTNK 000 05, PTGC 000 05 and subjected to freeze drying.

The disadvantage of this method is the insufficiently high yield of peroxidase, high labor intensity and duration of the process.

The closest is the way [US Pat. RF 2130070, C12N 9/08, 10.05.1999], in which the horseradish roots washed with water are cleaned by 1/3 of the mass in the presence of a 0.25% solution of food-grade ascorbic acid used as an extracting solution. Peroxidase from the purifications is extracted for 1 hour with a 0.25% solution of ascorbic acid, then the extract is filtered and centrifuged. To the supernatant, 5% sodium sulfite is added and kept for 24 hours at room temperature to "ripen" the enzyme. The "matured" enzyme solution is concentrated on ultrafiltration fiber apparatus with filters having a pore diameter of less than 40 kDa. Ammonium sulfate is added to a 10-fold concentrated solution to a final saturation of 85-90%, centrifuged, the precipitate is dissolved in a tenfold volume of bidistilled water and applied to a column filled with Sephadex G-25, elution is carried out with bidistilled water. Collect fractions containing peroxidase with an R Z value\u003e 0.1. Ammonium sulfate is added to the collected fractions until saturation 85-90%, centrifuged, the precipitate is dissolved in a 3-fold amount of bidistilled water and applied to a gel filtration column filled with Sephadex G-50, elution is carried out with bidistilled water. Collect fractions containing peroxidase, with an R Z value\u003e 0.5. The fractions are mixed, titrated to pH 4.4 and purified on carboxymethyl cellulose, the enzyme is eluted in a concentration gradient from 5 mM to 0.15 M acetate buffer (pH 4.4) (V \u003d S-500 ml, R-500 ml). Collect fractions with an R Z value\u003e 2.7 and with an enzyme concentration of at least 10 mg / ml. The fractions are combined, titrated to pH 5.0 and freeze-dried.

The disadvantages of this method are insufficiently high purity and activity and low yields of peroxidase.

The invention solves the problem of creating an industrial method for producing peroxidase from horseradish roots, which makes it possible to obtain peroxidase with high purity, high specific activity and high yield.

The problem is solved by a method of obtaining the peroxidase enzyme from horseradish roots, which includes grinding and homogenization of germinated horseradish roots, extraction of horseradish roots homogenizate, separation of ballast proteins and precipitation of peroxidase with ammonium sulfate salts, gel filtration of peroxidase solution on Sephadex, chromatographic purification of target peroxidase for carboxymethylcellulose , while the crushed horseradish roots are extracted with 0.15 ± 0.01 M sodium chloride solution with pH \u003d 4.4 ± 0.2; gel filtration of peroxidase solution is carried out on Sephadex G-100 with elution with 0.15-0.2 M sodium chloride solution with pH 4.4-5.0, dialysis against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0 ± 0.1 and concentration with additional purification on DEAE-cellulose with elution with the same buffer, followed by dialysis against deionized water.

Precipitation of proteins with salts of ammonium sulfate is used twice: 45-48% saturation is used to separate ballast proteins, and 85-90% saturation is used to precipitate peroxidase,

Chromatographic purification of peroxidase on carboxymethylcellulose is preceded by dialysis against a sodium acetate buffer with a pH of 4.4-5.0.

Freeze drying is preceded by dialysis of the peroxidase solution against deionized water.

Figure 1 shows a schematic diagram of the isolation of peroxidase from horseradish roots.

The horseradish roots are washed under running water and germinated for 140-160 hours at a temperature of + 25 ± 1 ° C. Sprouted roots are crushed and extracted with 0.15 M sodium chloride solution for 12 ± 2 h with constant stirring, then centrifuged.

To supernatant-1 (Fig. 1) with continuous stirring add 16.7 ± 0.05 kg (45-48% saturation) of ammonium sulfate, the formed precipitate is separated by centrifugation, and to the resulting supernatant-2 (Fig. 1) add 11 more , 8 ± 0.05 kg (85% saturation) of ammonium sulfate, the precipitate (Fig. 1) is separated by centrifugation and dissolved with distilled water to a final volume of 200 ± 10 ml. After centrifugation, the supernatant containing peroxidase is applied to a column packed with Sephadex G-100, and eluted with 0.15 M sodium chloride solution at a rate of 100 ml / h; 20 ml fractions are collected during the elution. The collected fractions are measured R Z \u003d D 408 / D 275. Fractions in which RZ is not less than 0.8 are combined and dialyzed against sodium acetate buffer (pH 4.4 ± 0.2) (buffer-1), then the desalted peroxidase solution is layered on a column packed with carboxymethylcellulose and equilibrated with buffer-1 , and eluted with a linear gradient of 5 mM - 0.1 M acetate buffer (pH 4.4 ± 0.2) (V \u003d S-0.5 L, R-0.5 L). In protein fractions, the R Z value is measured. Fractions in which the RZ value is not less than 2.5 are combined and dialyzed against potassium phosphate buffer (pH 8.0 ± 0.1) (buffer-2), the dialyzed enzyme solution is layered on a column packed with DEAE-cellulose and eluted buffer-2. Fractions in which the R Z value is not less than 3.0 are combined and dialyzed against deionized water, then transferred to a sterile heat-resistant flask, frozen with liquid nitrogen and lyophilized until the drug is completely dry.

The essential distinguishing features of the proposed method for producing biologically active substances are:

The use of gel filtration on Sephadex G-100 for the most complete purification of peroxidase from low molecular weight impurities;

Dialysis against sodium acetate buffer (pH 4.4-5.0), which allows to desalt the peroxidase solution and prepare it for chromatography on carboxymethyl cellulose;

Dialysis against potassium phosphate buffer (pH 8.0 ± 0.1), which makes it possible to transfer the peroxidase solution to the optimal buffer with the optimal pH for application to DEAE-cellulose;

Ion exchange chromatography of peroxidase on DEAE-cellulose as a technique that provides not only additional purification, but also the concentration of the enzyme solution.

Thus, we propose a new approach that allows for the processing of horseradish roots to obtain the peroxidase enzyme of high purity and specific activity.

The difference between the proposed method and the closest analogue and prototype is as follows.

In the claimed method, the crushed horseradish roots are extracted with 0.15 ± 0.01 M sodium chloride solution with pH \u003d 4.4 ± 0.2, thereby achieving the most complete transition of the enzyme into the solution, and the enzyme does not lose its activity.

In the analogue of the invention [US Pat. RF 2130070, C12N 9/08, 10.05.1999] peroxidase from purifications (without homogenization!) Is extracted for 1 hour with a 0.25% solution of food-grade ascorbic acid, which can affect the yield of peroxidase, since purifications are not homogenized, and therefore the enzyme goes into solution far from completely, while the extraction proceeds under acidic conditions, which can lead to partial inactivation of the enzyme. In the prototype of the invention [A.S. Bulgaria 46675, C12N 9/08, 15.02.90] the enzyme from the homogenized horseradish roots is extracted with water, which also leads to a low yield of peroxidase from the homogenized product into the solution.

In contrast to the analogue of the invention, where peroxidase is precipitated twice with 85-90% saturation with ammonium sulfate, and the prototype of the invention, where proteins are precipitated with ammonium sulfate salts four times, in the claimed method, ballast proteins are separated by 45-48% saturation, and then peroxidase 85 % saturation.

In the analogue and the prototype of the invention, the concentration is carried out by the method of ultrafiltration, while in the analogue of the invention, ultrafiltration precedes the ammonium sulfate precipitation of peroxidase, which leads to a high probability of coprecipitation of impurity proteins, and, accordingly, to a lower purity of the final product. In the claimed method, peroxidase is concentrated on the ion-exchange sorbent DEAE-cellulose at the final stage of the technological process, which leads to additional purification of the enzyme.

In the claimed method, gel filtration is carried out on Sephadex G-100 with elution with 0.15 ± 0.01 M sodium chloride solution (pH 4.4-5.0), which allows you to completely get rid of the greenish tint of the peroxidase solution due to the presence of chlorophyll and related compounds , and substances with smaller molecules than peroxidase. In an analogue of the invention, gel filtration is carried out on Sephadex G-25, which is inferior to Sephadex G-100 in terms of its ability to retain impurity particles, given the size of the peroxidase molecule (about 40 kDa).

The essence of the invention is illustrated by the following examples.

Example 1. Obtaining a crude extract

50 kg of horseradish roots are washed under running water and germinated for 140-160 hours at a temperature of + 25 ± 1 ° C. Sprouted roots are crushed on a paddle homogenizer and filled with 50 liters of 0.15-0.2 M sodium chloride solution (pH 4.4 ± 0.2). The suspension is extracted for 12 ± 2 h with constant stirring, then centrifuged.

Ammonium sulfate is added to supernatant-1 with a volume of 60 liters to separate ballast proteins with continuous stirring to 45-48% saturation. (By 100% saturation we mean the amount of salt, when added, the solution becomes saturated, and upon further addition of salt, the solution becomes supersaturated, and the salt precipitates. For solutions with ammonium sulfate, 100% saturation is the addition and complete dissolution 70, 7 g of ammonium sulfate in 100 ml of distilled water.) The precipitate-1 is separated by centrifugation. Next, the formed supernatant-2 with a volume of 55 l for peroxidase precipitation is saturated with ammonium sulfate to 85%, the precipitate-2 is separated by centrifugation. The formed precipitate-2 is dissolved with deionized water to a final volume of 200 ± 10 ml, the insoluble precipitate-3 is separated by centrifugation.

Gel chromatography on Sephadex G-100.

The peroxidase solution is applied to a 1 L column packed with Sephadex G-100. Elution is carried out with 0.15-0.2 M sodium chloride solution (pH 4.4-5.0) at a rate of 100 ml / h, during the elution 20 ml fractions are taken. In the collected fractions, R Z \u003d D 408 / D 275 is measured. Fractions in which R Z is not less than 0.8 are combined.

The collection container is placed 5 l of 5 ± 0.1 mM sodium acetate buffer (pH 4.4-5.0) (buffer-1) and a dialysis bag with combined peroxidase fractions. Dialysis is carried out with constant stirring for 24 hours, changing the buffer in the collection vessel three times.

The desalted enzyme solution is layered on a 1 L column packed with carboxymethyl cellulose and equilibrated with buffer-1. Elution is carried out at a rate of 50 ml / h in a linear gradient of 5 mM-0.1 M acetate buffer (pH 4.4 ± 0.2) (V \u003d S-0.5 L, R-0.5 L). In protein fractions, the R Z value is measured. Fractions in which the R Z value is not less than 2.5 are combined.

5 l of 20 ± 0.1 mM potassium-phosphate buffer (pH 8.0 ± 0.1) (buffer-2) and a dialysis bag with combined enzyme fractions are placed in the collection container. Dialysis is similar.

Concentration chromatography on DEAE-cellulose.

The enzyme solution with pH 8.0 ± 0.1 is layered on a 300 ml column packed with DEAE cellulose. Elution is carried out with buffer-2 at a rate of 50 ml / h. Fractions in which the R Z value is not less than 3.0 are combined.

The collection container is placed 5 l of deionized water and a dialysis bag with a solution of peroxidase. Dialysis is similar.

Freeze drying.

The peroxidase solution is transferred into a sterile heat-resistant flask, frozen with liquid nitrogen and lyophilized until the preparation is completely dry.

Example 2 (comparative)

In this example, the conditions for obtaining peroxidase according to the prototype were maintained, but to improve the basic parameters of peroxidase, two existing stages in the prototype were changed, namely: for ultrafiltration concentration of a peroxidase solution, two types of columns with hollow fibers are used, in contrast to the prototype, where one type of fibers is used, and fractional fractionation of proteins with ammonium sulfate salts (as in the claimed method).

Getting a coarse extract.

50 kg of horseradish roots are washed under running water and cleaned by 1/3 of the mass in the presence of 33.5 l of a 0.25% solution of food-grade ascorbic acid, in which the resulting cleaning is kept for 1 hour to extract the peroxidase enzyme. Next, the extract is centrifuged in a flow-through centrifuge and incubated for 24 hours to "ripen" the enzyme.

Primary purification and concentration.

1.7 kg (5% by weight) of sodium sulfite are added to the obtained extract, and the extract is subjected to concentration and primary purification by ultrafiltration in an installation with hollow polymer fibers UPV-6, equipped with columns with filters having pore sizes of 60 kDa and 5 kDa. A schematic diagram of the installation is shown in Fig. 2, which shows a centrifugal pump 1; prefilter 2; column with fibers having a pore size of 60 kDa 3; column with fibers having a pore size of 5 kDa 4; peroxidase filtrate 5; concentrated solution of peroxidase 6; filtrate containing low molecular weight proteins 7.

Fractionation of proteins with ammonium sulfate salts.

Ammonium sulfate is added to a 6 liter concentrate to separate ballast proteins with continuous stirring to 45-48% saturation. The precipitate is separated by centrifugation. Next, the resulting supernatant with a volume of 5.7 l for peroxidase precipitation is saturated with ammonium sulfate to 85%, the precipitate is separated by centrifugation, which is dissolved with bidistilled water to a final volume of 200 ± 10 ml, the undissolved precipitate is separated by centrifugation.

Gel chromatography on Sephadex G-25 and G-50.

Further purification of peroxidase is carried out on a gel filtration column filled with Sephadex G-25 and equilibrated bidistilled water. The peroxidase solution is applied to the column, elution is carried out with bidistilled water. Collect fractions in which R Z is not less than 0.2. To the collected fractions add ammonium sulfate to 90% saturation, stir until the salt is completely dissolved and centrifuged. The precipitate is dissolved in a 3-fold by volume amount of bidistilled water and applied to a gel filtration column filled with Sephadex G-50 and equilibrated with bidistilled water. Elution is carried out with bidistilled water. Collect fractions in which R Z is not less than 0.6. The peroxidase fractions are adjusted to pH 4.4 with 50% acetic acid.

Chromatography on carboxymethyl cellulose.

Peroxidase fractions are layered on a 1 L column packed with carboxymethyl cellulose pre-equilibrated with 5 mM acetate buffer pH 4.4. Elution is carried out with a linear gradient of 5 mM-0.15 M acetate buffer (pH 4.4 ± 0.2) (V \u003d S-0.5 L, R-0.5 L) for 1 hour. In protein fractions, the R Z value is measured. Fractions in which the R Z value is not less than 2.7 are combined, adjusted to pH 5.0 by adding ammonia and lyophilized.

Comparative data for example 1 and 2 are shown in the table.

Table Comparative data of the main quality parameters of horseradish peroxidase using two methods of obtaining.
The name of the technological parameter, units of measurement.	Example 1 (claimed method)	Example 2 (known method)
Yield by weight of peroxidase, g / kg of horseradish roots.	2,52-3,50	0,21-0,85
Specific activity of the preparation, EA / mg protein *	640-700	560-610
Spectrophotometer purity R Z \u003d D 408 / D 275	3,00-3,20	2,70-3,00
* - Specific activity was calculated using the data given in the work STP 103.34-83. Rules for calculating and processing the results of quantitative analysis. NIKTI BAV, 1983.

Thus, as can be seen from the examples and the table, the claimed method for producing peroxidase from horseradish roots (example 1) allows to obtain peroxidase in high yields, with a purity and activity sufficient for the use of this enzyme as a component of conjugates for immunoassays. And in the conditions of the prototype (example 2), even with the improvement of two stages that improve the performance, yield, specific activity and purity of the target peroxidase below those of the proposed method.

This method can be used in the industrial production of peroxidase from horseradish roots.

CLAIM

1. A method of obtaining the enzyme peroxidase from horseradish roots, including crushing and homogenization of germinated horseradish roots, extraction of homogenized horseradish roots, separation of ballast proteins and precipitation of peroxidase with ammonium sulfate salts, gel filtration of peroxidase solution on Sephadex, chromatographic purification on carboxymethylcellulose target, lymphocylcellulose the fact that the crushed horseradish roots are extracted with 0.15 ± 0.01 M sodium chloride solution; gel filtration of the peroxidase solution is carried out on Sephadex G-100, dialysis against sodium acetate buffer with pH 4.4-5.0 and dialysis against potassium phosphate buffer with pH 8.0 ± 0.1, and concentration with additional purification on DEAE -cellulose with elution with the same buffer, followed by dialysis against deionized water.

2. The method according to claim 1, characterized in that the crushed horseradish roots are extracted with 0.15 ± 0.01 M sodium chloride solution with pH 4.4 ± 0.2.

3. The method according to claim 1, characterized in that the precipitation of proteins with ammonium sulfate salts is used twice: 45-48% saturation is used to separate ballast proteins, and 85-90% saturation is used to precipitate peroxidase.

4. The method according to claim 1, characterized in that gel filtration is performed on Sephadex G-100 with elution with 0.15-0.2 M sodium chloride solution with pH 4.4-5.0.

5. The method according to claim 1, characterized in that the chromatographic purification of peroxidase on carboxymethyl cellulose is preceded by dialysis against a sodium acetate buffer with a pH of 4.4-5.0.

6. A method according to claim 1, characterized in that freeze drying is preceded by dialysis of the peroxidase solution against deionized water.

About 44,173.9 Da, is a glycoprotein and has four lysine amino acid residues to bind to the molecule to be labeled.

The product of horseradish peroxidase activity is a colored or luminescent compound suitable for detection and quantitation. HRP is often used in conjugates to detect specific molecules. For example, in the case of Western blotting, HRP conjugates with antibodies against the desired proteins or molecules are used; in this case, the antibody has specificity for a given target, and the HRP generates a detectable signal. ... Horseradish peroxidase is also used in techniques such as ELISA and for immunohistochemical analysis.

Horseradish peroxidase is an ideal enzyme for many techniques because it is relatively small, relatively stable, and cheaper than alternatives such as alkaline phosphatase. HRP has used aboutthe greatest number of revolutions per unit of time and therefore ensures the development of a sufficiently strong signal in a relatively short period of time.

Horseradish peroxidase in free form or in the form of conjugates with other molecules requires a substrate for imaging. HRP oxidizes the substrate in the presence of hydrogen peroxide to form products that can be detected spectrophotometrically.

Commercially available horseradish peroxidase substrates 3,3 ', 5,5'-Tetramethylbenzidine (rus. TMB) and 3,3 "-diaminobenzidine (eng. DAB) upon oxidation give colored products, and chemiluminescent substances SuperSignal, ECL are sources of detectable light under the action of HRP.

Enhanced chemiluminescence (ECL)

Horseradish peroxidase catalyzes the oxidation of luminol to 3-aminophthalate via a series of intermediates. This reaction is accompanied by a low intensity glow with a wavelength of 428 nm. In the presence of certain substances, it is possible to achieve an intensification of the glow up to a thousand times. The phenomenon of enhanced glow is called enhanced chemiluminescence (eng. enhanced chemiluminescence, ECL ). The most effective enhancers are phenol derivatives, for example, p-iodophenol. ECL detects about 0.5 picograms of nucleic acid in Southern blotting.

Notes

External links

Wikimedia Foundation. 2010.

See what "Horseradish peroxidase" is in other dictionaries:

horseradish peroxidase - - Topics in biotechnology EN horseradish peroxidase ... Technical translator's guide

- [[Image: | px | Thyroid peroxidase chemical structure]] thyroid peroxidase Legend Symbol (s) TPO Entrez… Wikipedia

Western blot analysis of proteins separated by gradient polyacrylamide gel electrophoresis in the presence of SDS. Western blotting (protein immunoblot, English Western blot) is an analytical method used to determine ... ... Wikipedia

Western blotting (Western blot, protein immunoblot, English Western blot) is an analytical method used to determine specific proteins in a sample. At the first stage, it uses polyacrylamide gel electrophoresis of proteins for ... ... Wikipedia

- (English Peroxiredoxins, Prxs, EC 1.11.1.15) a widespread family of antioxidant enzymes. In mammals, enzymes of this group control the level of cytokine-induced peroxides involved in the transmission of cellular signals. ... ... Wikipedia

Glutathione peroxidase (GP) is a family of enzymes that protect the body from oxidative damage. HPs catalyze the breakdown of lipid peroxidases and hydrogen peroxide. Several genes are known that encode different forms of glutathione peroxidases, differing ... ... Wikipedia

Peroxidase is one of the most common enzymes found in plants, microbes, and animal tissues. This enzyme catalyzes the oxidation of a wide range of organic compounds with hydrogen peroxide to form toxic peroxides that are removed from living organisms. Peroxidase is a glycoprotein consisting of a polypeptide chain that forms a two-domain globule and a heme prosthetic group with an iron atom located between the domains.

The main distinguishing feature in the structure of horseradish peroxidase C in comparison with other plant peroxidases is the presence of a 34 amino acid residue between the helices of phenylalanine (F) and glycine G residues (Fig. 1). This region, which is part of the channel for substrate access, is not found in peroxidases of other classes; moreover, it differs even within its own class. For horseradish peroxidase C, which is characterized by a larger F-G insert (by 7 amino acid residues), a key residue has been identified that can enter into direct interactions with aromatic donor molecules. Horseradish peroxidase isoenzyme C is unique in that it has a ring of three peripheral residues, Phe142, Phe68 and Phe179, which protects the approach to the exposed edge of the heme. This aromatic region is important for realizing the ability of peroxidase to bind aromatic substrates.

Figure: 1. Spatial structure of horseradish peroxidase C.

A feature of the processes of peroxidase catalysis is the formation of a number of spectrophotometrically distinguishable complexes. A simplified diagram of the peroxidase cycle is as follows:

E + H 2 O 2 → E 1; k 1

E 1 + AH 2 → E 2 + AH; k 2

E 2 + AH 2 → E + AH; k 3

where E, E 1, E 2 - respectively, the original peroxidase and its oxidized forms; AH 2, AH - respectively, the original substrate and its oxidized form.

The product of the first stage, E 1, formed by the action of hydrogen peroxide on the enzyme, is an oxidized peroxidase derivative containing two oxidative equivalents. In E 1, iron has a formal charge of +4. An additional oxidative equivalent in the E 1 molecule is localized either on the porphyrin macrocycle of peroxidase or on one of the functional groups of the enzyme.

Donor substrates can reduce E 1 directly to the native enzyme (two-electron reduction) or through the formation of an E 2 intermediate (one-electron reduction).

In the reaction of peroxidase oxidation, in addition to hydrogen peroxide, organic substrates - alkyl hydroperoxides, peroxybenzene acids, etc. - can act as an oxidizing agent (the first substrate). Peroxidase exhibits less specificity with respect to the second substrate; therefore, a number of electron donor compounds can be used as substrates for peroxidase and to be the basis of detecting systems in methods of analytical biochemistry and clinical medicine.