JPS5931519B2 - Extraction, separation and purification method of high purity protamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for easily extracting, separating, and purifying high-purity protamine.

Protamine is a strong basic protein with a relatively small molecular weight and high arginine content, which exists as nucleoprotamine bound to deoxyribonucleic acid in the sperm nuclei of salmon, trout, herring, mackerel, etc.

Protamine has long been used as a clinical drug in the form of long-acting insulin preparations, which are complexes with insulin, and protamine injections, which are antiheparin agents.At the same time, protamine has been used as a drug for separation and purification in biochemical research and the biochemical industry. Recently, it has become an indispensable substance as a stabilizer for certain enzymes and as an activator or auxiliary agent for anti-inflammatory substances. Conventionally, protamine was produced by grinding the mature milt of the fish in a meat grinder, suspending it in 4 to 5 times the volume of water, stirring thoroughly, and then filtering the emulsion through a cloth or wire mesh while stirring. Make litmus acidic with dilute acetic acid and centrifuge the aggregated sperm nuclei.

Furthermore, it is repeatedly washed with acetic acid water, and if necessary, it is degreased with alcohol and dried. Next, this sperm nucleus is extracted with dilute mineral acid for a certain period of time, the residue is removed by centrifugation, and the extraction of the residue is repeated.

Then, 2 to 3 times the volume of alcohol is added to the extract to separate it as protamine sulfate, or it is precipitated with sodium picrate as protamine pitalate, and further separated as protamine sulfate with sulfuric acid and alcohol. The most common purification method for protamine is repeated reprecipitation with alcohol as a sulfate salt, but as mentioned above, treatment via a picrate salt is said to be effective for purification, and there are other methods as well. General protein precipitants (platinum chloride, tungstic acid, metaphosphoric acid, ferrocyanic acid,
sulfosalcylic acid, etc.) and converting it into a sulfate salt by a conventional method, and separation and purification using an ion exchange resin, which has recently become common, are also carried out.

In any case, these conventional protamine extraction, separation, and purification methods are generally complicated and labor-intensive, or require expensive machinery and materials, making them unsuitable for mass production. Although it is appropriate, there is also a conflict between quality purity and yield, which is extremely disadvantageous economically.

The present inventors conducted research on methods to overcome the drawbacks of the conventional methods, and as a result, they found that they could easily extract high-purity protamine.
We have now completed the present invention, which enables separation and purification.

That is, the present invention involves directly extracting protamine-containing fish milt in its original form with dilute mineral acid without grinding it, and adding condensed phosphoric acid or its salt to the extract to precipitate it as a sparingly soluble protamine salt. This is separated as protamine mineral salt by double decomposition with highly concentrated inorganic salts, and after treating the acidic dilute aqueous solution with an adsorbent, it is purified by fractional precipitation with the minimum necessary amount or less of an organic solvent. This is a method for extracting, separating, and purifying high-purity protamine. The first feature of the present invention is the process of directly extracting the milt in its original form with dilute mineral acid without grinding it, and the second is the process of adding bound phosphoric acid or its salt to the extract. This is a process in which protamine mineral salts are separated by precipitating soluble protamine salts and double decomposing them with highly concentrated inorganic salts.The third step is to separate protamine mineral salts in an acidic dilute aqueous solution of protamine mineral salts, and then treating the protamine mineral salts with an adsorbent. , is a purification process in which precipitation is performed using the minimum necessary amount of organic solvent.

The present invention will be explained in detail below by dividing into the above three steps. Generally, the method for preparing the contents from animal tissue is (1)
) collecting tissue, (2) removing foreign tissue, (3)
Grind the tissue to make it easier to extract.

(4) Remove substances that interfere with extraction by washing with water or other solvents, (5) Extract the target component, (6)
This is achieved by separating only the target component as much as possible, and (7) refining to the target purity. The preparation of protamine from Milt is also only a special example of the above general method. As a matter of course, conventional methods for producing protamine are also carried out in accordance with the above-mentioned operations. That is, all conventional methods start from grinding the milt. However, it is extremely difficult to isolate sperm nuclei from an emulsion suspended in water by centrifugation or filtering. Even if auxiliary measures such as pre-heating or treatment with organic solvents are used, a high-performance centrifuge is used, or an effective filter aid is used, the expected effects will not be achieved. cannot be obtained. Separation and removal of the residue, which is fine particles after extraction, similarly involves a great deal of effort and difficulty. As long as repeated centrifugation steps are involved in this initial process, mass production is impossible, and this is the biggest difficulty in preparing protamine from milt. The first extraction step, which is a feature of the present invention, focuses on the uniqueness of milt in that the sperm nucleus, where protamine is located, is located in the sperm head, unlike general animal tissues, where the cell nucleus and cytoplasm are tightly integrated. This is an application of the fact that it does not necessarily require physical destruction to make it easier to extract.

In other words, dilute mineral acid is applied directly to the milt without grinding it, and only protamine is dissolved and extracted from the sperm head through acid penetration, and the milt membrane is used as if it were a dialysis membrane. Therefore, consideration was given to leaving most of the other tissues within Shirako. Of course, the acid-soluble components are extracted at the same time, but they can be removed later by another method, completely eliminating the effort of physically separating extremely fine sperm nuclei and other solid components in conventional methods. becomes. According to the method of the present invention, after extraction, the dehydrated, shrunken, and solidified milt are suspended in a colorless and transparent extract, so they can be completely separated simply by pouring out the extract. Extraction conditions such as the type and concentration of dilute mineral acid, extraction time and temperature can be freely selected as in conventional methods.

If complete extraction of protamine is desired in a short period of time, it is desirable to increase the acid concentration and circulate the liquid at room temperature or higher. The second separation step, which is a feature of the present invention, is a method for separating protamine in the extract, which uses a large amount of alcohol as in the conventional method, or a special precipitant such as sodium picrate. The method is to utilize condensed phosphoric acid or its salts, which are used in large quantities on a daily basis as an extremely common food additive, to separate them using a sparingly soluble protamine salt, and at the same time to utilize a partial purification effect. . Condensed phosphoric acid and its salts are generally known as protein precipitants, and an example of their use in the separation and purification of protamine is found in US Pat. No. 2,497,858.

However, the reason why this method has not become widely popular is that there were difficulties in replacing the condensed phosphate of protamine with a normal mineral salt. Incidentally, U.S. Patent No. 24978
In No. 54, protamine metaphosphate is heated and dissolved in 1N sulfuric acid, and then boiled until it is completely converted to sulfate. Naturally, as a result of the conditions involved in protein hydrolysis, the resulting protamine is inevitably damaged in some way, and for this reason its adoption was discouraged. The present invention was adopted based on the new finding that protamine mineral salts can be easily obtained by metathesizing protamine condensed phosphates with high concentration inorganic salts.

In other words, although protamine condensed phosphate dissolves even in 1N sulfuric acid only after heating and boiling, it easily dissolves in highly concentrated inorganic salts, leaving it in a state that is almost completely separated into protamine and condensed phosphoric acid. Protamine mineral salts can be separated into an oily state by simple operations such as appropriate salt concentration and temperature changes, and can be isolated using only liquid separation operations. As the condensed phosphoric acid and its salts, any of metaphosphoric acid, polyphosphoric acid, and salts thereof can be used. Further, inorganic salts for metathesis such as alkali salts, alkaline earth salts, and ammonium salts of mineral acids can be selected depending on the purpose. The solubility of protamine condensed phosphate is maximum when the inorganic salt concentration is about 2 mol, but it can be freely selected from 0.5 mol to a saturated solution depending on the relationship with liquid volume and temperature. Further, the dissolution temperature is appropriately selected depending on the salt concentration and the liquid amount, and the key is to select conditions that allow protamine to be obtained in the form of an oil based on the temperature difference. If a sulfate is used, protamine sulfate will separate into an oily state, and if it is dehydrated and solidified with alcohol or methanol, a white powder of protamine sulfate will be obtained. The third purification step, which is a feature of the present invention, is to treat the extracted and separated protamine mineral salt with an adsorbent in a dilute acidic aqueous solution, and then precipitate it fractionally with the minimum necessary amount of organic solvent to obtain a clinical drug. This is an extremely simple method for purifying high-purity protamine, which can be fully applied as a method for purifying protamine.

The extract from Milt contains proteins other than promemin, their decomposition products, nucleic acid decomposition products, and certain lipids and their decomposition products, which also adhere to the separated protamine mineral salts. However, the relative amounts of the above impurities do not necessarily exist uniformly depending on the raw material Milt, extraction conditions, separation conditions, etc. Therefore, targets are set for each impurity based on the principle of removing each impurity. Performing optimal purification processes is difficult in practice and requires a clever combination of multiple operations under greatest common denominator conditions.

In an acidic dilute aqueous solution of protamine mineral salt, the chemical bonds and physical adhesion with the impurity protamine mentioned above are almost completely broken or dissolved in a separated state, and substances other than protamine have a remarkable affinity for the adsorbent. I learned that most of them can be removed by using sexual characteristics.

Of course, some organic solvents are not adsorbed and naturally remain in the solution, so the amount of organic solvent used in the precipitation operation must be the minimum necessary amount. The concentration of the aqueous solution is preferably as dilute as possible, but it can be freely selected within the range of 1 to 10%.

The stronger the acidity, the better the solubility of impurities, usually PH3
The following are preferred. Any general adsorbent (eg, activated carbon, activated alumina, silica gel, bentonite, acid clay, diatomaceous earth, bone charcoal, etc.) can be used and can be appropriately selected depending on the main purpose.

The amount used may be about 1 to 10% in either case. The temperature conditions may be hot or cold, and the longer the treatment time, the better results will be obtained. The organic solvent may be any alcohol, acetone, etc., and the amount used is preferably at most the equivalent volume. As described above, the present invention comprises (1) extraction step, (2) separation step, (3)
It consists of three purification steps, and each step can be combined with conventional methods to achieve its own effects, but the best results are especially obtained when the three steps mentioned above are combined.

Examples are given below.

Example 1 Frozen salmon milt caught in Miyako, Iwate Prefecture in late November 1
Let 00k9 stand overnight, thaw it, and add 250f of 0.5N hydrochloric acid.
! was added and extracted for 5 hours at room temperature while circulating the liquid.

The separated extract was adjusted to pH 3-4 with aqueous ammonia,
To this was added 2.3 kg of an aqueous solution of sodium metaphosphate with stirring, and the mixture was left overnight. The supernatant was decanted, and 7.5 kg of candy-like protamine metaphosphate was dissolved in 2 mol ammonium sulfate of 151 with heating and allowed to stand overnight. The lower layer of oily protamine sulfate is dehydrated and solidified with alcohol, and dried to obtain 4.5 kg of white powder of protamine sulfate. Example 2 In the same manner as in Example 1, 1 kg of promemine sulfate obtained from herring milt was dissolved under heating in 201 of water, and purified with dilute sulfuric acid.
Hl. Adjust to 5.

507 activated carbon was added and stirred for 1 hour, then drained.

Stir into a colorless and transparent protamine sulfate aqueous solution\51? Add methanol and leave overnight. The supernatant liquid was decanted, and the candy-like protamine sulfate was dehydrated and solidified with 5' methanol and dried. Pure white high purity protamine sulfate powder 850y
is obtained. Analysis results A. Amino acid composition mol% indicates the mole percentage of each amino acid relative to the number of moles of all amino acids.

B. ultraviolet absorption

Claims (1)

[Claims] 1 Extract the fish milt containing protamine directly with dilute mineral acid without grinding it, add condensed phosphoric acid or its salt to the extract, precipitate it as a sparingly soluble protamine salt, and add it to the extract. High-purity protamine is purified by separating it as protamine mineral salt by double decomposition with concentrated inorganic salts, treating the acidic dilute aqueous solution with an adsorbent, and then precipitating it fractionally with the minimum necessary amount of organic solvent. Extraction, separation and purification methods.