JPS5931518B2 - How to extract protamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for easily extracting protamine from fish milt containing 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 has been 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 clumped 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 volumes of alcohol is added to the extract to separate it as protamine sulfate, or it is precipitated with sodium picrate as protamine picrate, 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 production methods are generally complicated and labor-intensive, or require expensive machinery and materials, making them unsuitable for mass production. However, there is also a conflict between quality purity and yield, which is extremely disadvantageous economically.

J As a result of intensive research into an extraction method suitable for producing protamine on an industrial scale, the present inventors found that by directly extracting the milt in its original form with a dilute mineral acid without grinding it, We have discovered that protamine can be extracted extremely easily and have completed the present invention.

That is, the present invention is a method for extracting protamine, which is characterized by directly extracting protamine-containing fish milt in its original form with dilute mineral acid without grinding it. Generally, the method for preparing the contents from animal tissue is as follows:
(1) Harvesting 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) as much as possible. This is achieved through the following operations: separating only the target component and (7) purifying it 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 labor 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. In the present invention, we focused on the uniqueness of milt in that the sperm nucleus, where protamine is located, is located in the sperm head, and extracted it by physical destruction, like in general animal tissue, where the cell nucleus and cytoplasm are tightly integrated. This is an application of the fact that an operation to make it easier is not necessarily necessary for extraction.

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 a separate method, and the traditional method of physically separating extremely fine sperm nuclei and other solid components is completely unnecessary. 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. Protamine extracted by the method according to the present invention can be further separated and purified by conventional methods such as those described above. Preferably, the following separation and purification method discovered by the present inventors is advantageous in industrial scale production.

That is, condensed phosphoric acid or its salt is added to the extract to precipitate a sparingly soluble protamine salt, which is separated as a protamine mineral salt by metathesis with highly concentrated inorganic salts, and the acidic dilute aqueous solution is treated with an adsorbent. After that, it is purified by fractional precipitation using the minimum necessary amount of organic solvent.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Frozen salmon milt caught in Miyako, Iwate Prefecture in late November 1
00kg was left overnight to thaw, and 0.5N hydrochloric acid 2501
is added and extracted for 5 hours at room temperature while the liquid is being circulated, and then the extract is poured out and separated from the dehydrated, shrunken, and solidified milt to obtain a colorless and transparent protamine extract.

For reference, an example of separating protamine from the protamine extract is as follows: The protamine extract is adjusted to pH 3 to 4 with aqueous ammonia, and an aqueous solution of 2.3k9 sodium metaphosphate is added to this while stirring. Add and leave overnight.

Claims (1)

[Claims] 1. A method for extracting protamine, which is characterized by directly extracting protamine-containing fish milt in its original form with dilute mineral acid without grinding it.