JPS5949238B2 - How to separate protamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for easily separating protamine mineral salts from a protamine extract obtained from milt containing protamine.

Protamine is a strong basic protein with a relatively small molecular weight and high arginine content, which exists as nucleobrotamine 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 antihebarin drugs. Although it is also used as a medicine, it has recently 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. Litmus acidify 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, add 2 to 3 times the volume of alcohol to the extract to separate it as protamine sulfate, or precipitate it as protamine picrate with sodium picrate, and then separate it as protamine sulfate with sulfuric acid and alcohol, or In addition, it is common to separate with other general protein precipitants (platinum chloride, tungstic acid, metaphosphoric acid, ferrocyanic acid, sulfosalcylic acid, etc.) and convert it into a sulfate by a conventional method. The most common method for purifying protamine is to repeat the reprecipitation with alcohol as a sulfate, but separation and purification using ion exchange resins, which have recently become commonplace, is also carried out.

In any case, these conventional protamine production methods are
In general, operations are complicated and require a lot of labor, or expensive machinery and materials are required, making them unsuitable for mass production, and there is also a conflict between quality purity and yield. It is also extremely disadvantageous economically.

The present invention solves the complicated operations, economical disadvantages, and poor yields of the method for separating protamine from a protamine extract among these conventional protamine production methods.

The feature of the present invention is that condensed phosphoric acid or its salt is added to a protamine extract obtained from fish milt containing protamine to precipitate a sparingly soluble protamine salt, and this is double decomposed with a high concentration inorganic salt to produce protamine mineral acid. In the method of separating salt.

The method according to the present invention will be explained in detail below. The conventional method of adding a large amount (approximately 3 times the volume) of alcohol to the protamine extract to precipitate and separate protamine mineral salts is not only economically disadvantageous, but also contains impurities other than protamine. Co-precipitation is required, which also has the disadvantage of complicating purification operations.

In addition, when precipitated with sodium picrate, 6
Picric acid is removed with dilute hydrochloric acid and ether in a 7% acetone aqueous solution, and then separated as protamine sulfate with sulfuric acid and alcohol, but it is extremely difficult to completely remove traces of picric acid, which has a characteristic yellow color. In some cases, this results in a significant decrease in yield. The use of other general protein precipitants has generally similar drawbacks. In the present invention, we utilize condensed phosphoric acid or its salt, which is used in large quantities daily as an extremely common food additive.
In addition to separating using a sparingly soluble protamine salt, it also makes use of some purification effects.

Condensed phosphorus precipitates and their 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 three flaws in the method of replacing the condensed phosphate of protamine with a common mineral salt. Incidentally, U.S. Patent No. 2497
854 relies on the procedure of heating and dissolving protamine metaphosphate in 1N sulfuric acid, boiling it until it is completely converted to sulfate, and then adding alcohol or acetone to obtain protamine sulfate. ．． That is. Naturally, as a result of the conditions for protein hydrolysis, the resulting protamine may be damaged in some way, and for this reason, its use was discouraged. The method of the present invention was adopted based on the new finding that protamine mineral salts can be easily obtained by double decomposing protamine condensed phosphates with high-concentration φ-concentration inorganic salts.

Although protamine condensed phosphate dissolves only in 1N sulfuric acid by heating and boiling, it easily dissolves in high concentration inorganic salts, and is completely dissociated 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 or 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 moles, but 0.
The concentration can be freely selected from 5 mol to a concentration closer to a saturated solution depending on the relationship with the liquid volume and temperature. In addition, the dissolution temperature can be adjusted as appropriate depending on the salt concentration and liquid volume, 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 acetone, a white powder of protamine sulfate will be obtained. Although the protamine extract used in the present invention can be obtained by the conventional method as described above, it is preferably obtained by the following method discovered by the present inventors. This is a method for extracting protamine-containing fish milt by directly exposing it to dilute mineral acid in the form of X without grinding it.

Furthermore, the protamine separated according to the present invention can be further purified by conventional methods such as those described above.

As a method suitable for industrial mass production, the following method discovered by the present inventors is preferred. That is, after treating an acidic dilute aqueous solution of protamine mineral salt obtained by separating it from a protamine extract with an adsorbent,
This is a method of fractional precipitation using the minimum amount of organic solvent required or less. Examples and reference examples are given below.

Reference example: Frozen salmon milt 1 caught in Miyako, Iwate Prefecture in late November
Leave 0.0kg overnight to thaw, add 0.5N hydrochloric acid 250
1 and extracted for 5 hours at room temperature while circulating the liquid to obtain a protamine extract.

EXAMPLE The protamine extract obtained in the above reference example was adjusted to a PFI of 3 to 4 with aqueous ammonia, and 2.3 kg of an aqueous solution of sodium metaphosphate was added thereto with stirring and left overnight.

Decant the supernatant to obtain candy-like protamine metaphosphate 7.51
<g is dissolved in 1.51 ammonium sulfate with heating and left overnight. The extracted protamine sulfate in the lower layer is separated, dehydrated with alcohol, solidified, and dried to obtain 4.5 kg of white powder of protamine sulfate.

Claims (1)

[Claims] 1. Condensed phosphoric acid or its salt is added to a protamine extract obtained from fish milt containing protamine to precipitate a sparingly soluble protamine salt, which is double decomposed with high concentration inorganic salts to produce a protamine mineral salt. A method for separating protamine.