JPH0699477B2 - Method for purifying protamine mineral acid salt - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying protamine mineral acid salt.

2. Description of the Related Art Protamine is a relatively small basic protein having a molecular weight (about 4000 to about 10000) and exists as a nuclear protein bound to a nucleic acid (DNA) in sperm of fish such as herring, salmon and trout.

Protamine salts, especially their sulfates, are known to inhibit heparin from coagulating blood, to maintain insulin's medicinal effect, to stabilize enzymes or to stabilize antibiotics, and the like. Is used for various medicines.

As a method for purifying the protamine mineral salt obtained by extraction and separation from fish shirako, for example, it is disclosed in JP-B-49-24209, JP-A-55-2612 or JP-B-59-31519. ing.

The technique described in Japanese Patent Publication No. Sho 49-24209 is a purification method in which trichloroacetic acid is added to the extract of fish algae to remove impurities other than protamine as insoluble substances. This method requires an operation of removing insoluble substances and an operation of separating dissolved protamine mineral acid salt, and is a very complicated process. Further, since the protamine mineral acid salt obtained by this method has poor purity, another purification step is required.

Also, there is no teaching in the present method to utilize the solubility of protamine mineral salts for purification.

The technology described in JP-A-55-2612 and JP-B-59-31519 is a method in which an acidic dilute aqueous solution of protamine mineral salt obtained by extraction and separation from fish shirako is treated with an adsorbent, and then an organic solvent is used. This is a purification method that separates and precipitates by.

The method removes impurities other than protamine mineral acid with an adsorbent such as activated carbon, and teaches nothing about purification using the solubility of protamine mineral acid salt.

Problems to be Solved by the Invention As a result of studying a purification method for obtaining protamine sulfate with higher purity and high yield, the present inventors have found that protamine sulfate and contaminant proteins are different in solutions having different inorganic salt concentrations. It was discovered that the solubilities differ markedly. By utilizing this new finding, it has been possible to solve all of the above-mentioned problems and to complete a method capable of easily and efficiently obtaining a highly pure protamine sulfate.

MEANS FOR SOLVING THE PROBLEMS The present invention is to dissolve protamine mineral salt obtained by extracting and separating from fish spores in warm water, and adding 0.05 to 0.2 mol of sulfate to water in 1 liter of warm water. The present invention relates to a method for purifying a protamine mineral salt, which comprises depositing and cooling the protamine mineral acid salt.

The protamine mineral acid salt that can be used in the present invention may be any protamine mineral acid salt obtained by extracting and separating from fish, for example, herring, salmon, trout or mackerel, etc., by a known method, and is not particularly limited. Not something.

In the present invention, it is preferable to use protamine sulfate as the protamine mineral acid salt.

The higher the concentration of protamine mineral salt dissolved in warm water, the better.

The warm water may be at a temperature at which the protamine mineral salt to be dissolved is dissolved.

The sulfate that can be used in the present invention is ammonium sulfate, sodium sulfate or the like.

The sulfate salt may be dissolved in warm water together with the protamine mineral acid salt, or may be added after adding the protamine mineral acid salt.

Sulfate is added in an amount of 0.05 to 0.2 mol per 1 liter of warm water. If it is less than 0.05 mol or more than 0.2 mol, the protamine mineral acid salt cannot be efficiently precipitated after cooling the warm water.

The warm water is cooled to 10 ° C or lower, preferably 5 ° C or lower. 10 ° C
If it is higher, the protamine mineral salt cannot be efficiently precipitated.

It is not necessary to adjust the pH of hot water. When adjusting,
After adding protamine minerals and before adding sulfate
A range of 7 is desirable.

By performing purification according to the present invention, it is possible to remove salts, acids and contaminating proteins adhering to the precipitate of protamine mineral acid salt during extraction and separation of Miko.

According to the present invention, the crude protamine mineral salt extracted and separated from the white larva of fish can be efficiently purified by a simple means such as recrystallization.

In the present invention, the solubility of protamine mineral salt in solution 1 is 0.05 to 0.6 of ammonium sulfate or sodium sulfate.
It is based on the new finding that it is minimized in a solution in a very low concentration range which is dissolved in a molar state.

Table 1 shows the relationship between the solubility (% by weight) and the sulfate concentration of protamine sulfate in an aqueous solution having a water temperature of 5 ° C. and pH 2.5. In the table, the concentration represents the number of moles dissolved in Solution 1.

From Table 1, the solubility of protamine sulphate is such that the salt concentration is 0.05 to 0.
It can be seen that it becomes the minimum in the range of 2 moles.

The present invention will be described in more detail with reference to examples.

Example 1 Extraction Frozen salmon shirako was thawed, washed with water, drained and ground.

100 g of ground salmon Shirako was added to 500 ml of 5% (1N) sulfuric acid, and the mixture was stirred at room temperature (24 ° C) for 1 hour. After stirring, the residue was removed by centrifugation to obtain 550 ml of protamine sulfate extract.

Separation To the extract, 30 ml of concentrated ammonia water was added at room temperature of 24 ° C. with stirring to adjust the pH to 3, and 124 g of ammonium sulfate was added little by little to precipitate contaminating proteins. The contaminant protein precipitate was removed by centrifugation to obtain 630 ml of a supernatant.

While stirring, 130 g of ammonium sulfate was gradually added to the supernatant at room temperature of 24 ° C. to salt out protamine sulfate. The salted-out protamine sulfate was separated by centrifugation to obtain 10.4 g of white candy-like protamine sulfate.

Purification Further, 10.4 g of white candy-like protamine sulfate obtained as described above was dissolved in 100 ml of warm water (40 to 50 ° C.), and the concentration of ammonium sulfate was 0.09 mol per liter of the solution. Then, the solution was cooled to 5 ° C. and left at that temperature for 2 days to precipitate protamine sulfate.

The precipitated supernatant of protamine sulfate was decanted off to obtain a transparent oily protamine sulfate. It was freeze-dried to obtain 4.76 g of white protamine sulfate (yield 4.76%).

Example 2 Extraction Step Frozen salmon Shiroko was thawed, washed with water, drained and ground.

1 kg of ground salmon Shirako and 2.35 mol of ammonium sulfate were added to 2 l of 10% (2N) sulfuric acid and stirred at 30 ° C for 1 hour.
After stirring, the residue was removed by centrifugation to obtain 2.5 liters of protamine sulfate extract.

53.3 g (2.13% by weight) of protamine sulfate in the extract
It was included (dry conversion).

Separation step 2.5 l of the extract was cooled to 5 ° C. and allowed to stand at that temperature for 16 hours to separate a protamine sulfate precipitate and a supernatant. The supernatant was decanted and collected.

The protamine sulfate precipitate was white candy-like, and the yield was 102 g.

The recovered supernatant was 2.43 liters. Component is sulfuric acid
The content was 7.26%, ammonium sulfate 1.72 mol / liter, contaminating protein 1.6% by weight, and protamine sulfate 0.4% by weight (9.72 g).

Purification step 102 g of protamine sulfate obtained in the separation step was dissolved in 800 ml of warm water (40 to 50 ° C), and about 0.14 per liter of the solution was dissolved.
The concentration was molar ammonium sulfate. Then add the solution to 5
The mixture was cooled to 0 ° C. and allowed to stand overnight at that temperature to separate a protamine sulfate precipitate and a supernatant.

The supernatant liquid was decanted and removed to obtain a transparent oily protamine sulfate. Lyophilize it and white Protamine Sulfate 43.2
g was obtained (yield 4.32%).

Comparative Example 1 Example 1 was repeated except that the purification step in Example 1 was not performed. The obtained white candy-like protamine sulfate was lyophilized to obtain 6.30 g (yield 6.30%) of white protamine sulfate powder.

Comparative Example 2 Example 2 except that the purification step in Example 2 was not performed.
Same as above. The obtained white candy-like protamine sulfate was freeze-dried, and 53.2 g of white protamine sulfate powder was obtained.
(Yield 5.32%) was obtained.

Example 3 Protamine sulfate obtained in Examples 1 and 2 and Comparative Examples 1 and 2 was used as a sample, and its purity was measured.

The purity was examined by chromatography, absorption spectrometry, the solubility of the solution when protamine sulfate was dissolved in water, and the antibacterial activity.

High performance liquid chromatography was used for the chromatography. The purity of the sample was determined by using Wako Pure Chemical Industries, Ltd. Protamine Sulfate (salmon) as the standard, and comparing it with the purity of 1.00.
Shown as purity. In addition, the product obtained by multiplying the HPLC purity by the sample yield is shown as the standard product yield. Here, the standardized yield refers to the yield when the amount of protamine sulfate obtained from 100 g of white roe is converted to the amount of protamine sulfate having a purity of about the standard, and is considered as a scale for comparison of separation efficiency of protamine sulfate. Good. The results are shown in Table 2.

Comparing the HLPC purities, it can be seen that the purity of protamine sulfate obtained according to the present invention is about 1.2 times better than that of the comparative example.

For antibacterial activity, protamine sulfate (salmon) manufactured by Wako Pure Chemical Industries, Ltd. was used as a standard, and B. subtilis ( B.su
The antibacterial activity against btilis IAM 1069 strain) was compared as 1.00 and shown as the antibacterial activity specific activity. In addition, the product obtained by multiplying the specific activity of antibacterial activity and the yield of the sample is shown as the standardized active yield. The standardized active yield refers to the yield when converted to the amount of the standard required to show the same antibacterial activity as the amount of protamine sulfate obtained from 100 g of Shiroko.Comparison of separation efficiency of protamine sulfate You can think of it as a measure of. The results are shown in Table 2. When comparing the antibacterial activity specific activity, protamine sulfate obtained according to the present invention is about 1.
You can see that it is twice as good.

The absorption spectrometry measured the absorbance at 260 nm attributed to nucleic acid and 280 nm attributed to contaminant protein. The measurement was carried out at a temperature of 20 ° C. in an aqueous solution containing 0.1% by weight of the sample. The results are shown in Table 3. Pure protamine sulfate does not absorb light above 220 nm. As can be seen from Table 3, the protamine sulfate obtained according to the present invention has a much smaller amount of contaminating proteins than the protamine sulfate obtained according to the comparative example.

The visual observation of the solution state was performed with a 2% aqueous solution (20 ° C.) of each sample. Both protamine sulfate obtained according to the present invention and the comparative example were completely dissolved, and the presence of insoluble matter was not recognized.

EFFECTS OF THE INVENTION According to the present invention, crude protamine mineral acid salt obtained from fish spores can be purified by a simple process without loss.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Japanese Patent Publication Sho 49-24209 (JP, B1) Japanese Patent Publication Sho 59-31518 (JP, B2)

Claims (1)

[Claims] 1. A protamine mineral acid salt obtained by extraction and separation from fish shirako is dissolved in warm water, and then 0.05 to 0.2 mol of a sulfate salt is added to 1 liter of the warm water, followed by cooling. Method for purifying protamine mineral salt.