JPS62187492A - Method for purifying protamine mineral acid salt - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as an anticoagulant, etc., in high purity, simply and efficiently, by dissolving a protamine mineral acid salt obtained from milt of fish in warm water, adding a small amount of a sulfate to the warm solution in water and cooling. CONSTITUTION:Firstly, a protamine mineral acid salt is extracted from milt of fish by a well-known method, separated and dissolved in warm water usually at 40-50 deg.C. Then the warm solution in water is blended with a small amount of a sulfate, preferably 0.005-0.2mol based on 1l warm solution of the sulfate and cooled to precipitate the aimed compound. The warm solution is preferably cooled to <=5 deg.C.

Description

[Detailed description of the invention] Fields of use on the 1st name The present invention relates to a method for purifying protamine mineral salts.

Prior art protamine has molecules ff1 (about 4000 to about 10000)
It is a relatively small basic protein that exists as a nuclear protein bound to nucleic acid (DNA) in the sperm of fish such as herring, salmon, and trout.

Protamine salts, especially their sulfates, are known to have the effect of inhibiting heparin's blood clotting, prolonging the efficacy of insulin, stabilizing enzymes, and stabilizing antibiotics, and their properties. It is used as a PI in various pharmaceutical products.

Methods for purifying protamine mineral salts obtained by extraction and separation from fish milt are described, for example, in Japanese Patent Publication No. 49-24209, Japanese Patent Application Laid-Open No. 55-2612, or Japanese Patent Publication No. 59-1989.
It is disclosed in Japanese Patent No.-31519 and the like.

The technique described in Japanese Patent Publication No. 49-24209 is a purification method in which trichloroacetic acid is added to a fish milt extract to remove impurities other than protamine as insoluble substances. This method requires operations to remove insoluble substances and to separate dissolved protamine mineral salts, and is comprised of very complicated steps. Furthermore, since the protamine mineral salt obtained by this method has poor purity, another purification step is required.

Furthermore, this method does not teach anything about purification using the solubility of protamine mineral salts.

Japanese Unexamined Patent Publication No. 55-2612 and Japanese Patent Publication No. 59-315
The technique described in Publication No. 19 is a purification method in which an acidic dilute aqueous solution of protamine mineral salt extracted and separated from fish milt is treated with an adsorbent, and then fractionated and precipitated with an organic solvent.

This method involves removing impurities other than the protamine mineral salt using an adsorbent such as activated carbon, and does not teach anything about purifying the protamine mineral salt by utilizing its solubility.

Problems to be Solved by the Invention The present inventors have investigated purification methods for obtaining protamine sulfate with higher purity and higher yield. As a result, protamine sulfate and contaminant proteins can be separated from solutions with different concentrations of inorganic salts. It was discovered that the solubility was significantly different. By utilizing this new knowledge, all of the above problems can be solved,
We have completed a method to easily and efficiently obtain highly pure protamine sulfate.

Seven Steps to Solve the Problems The present invention involves dissolving protamine mineral salt obtained by extraction and separation from fish milt in hot water, then adding a small amount of sulfate to the hot water and cooling it to dissolve protamine. The present invention relates to a method for purifying protamine mineral salts, which is characterized by precipitating the mineral salts.

The protamine mineral salt used in the present invention may be any protamine mineral salt obtained by extraction and separation from fish such as herring, salmon, or milt of mackerel by a known method, and is not particularly limited. It's not a thing.

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

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

The hot water may have a temperature that allows the protamine mineral salt to be dissolved.

Sulfates that can be used in the present invention include ammonium sulfate and sodium sulfate.

The sulfate may be dissolved in hot water together with the protamine mineral salt, or may be added after the protamine mineral salt is added.

Sulfate is added in an amount of 0.05 to 0.2 mol per liter of hot water. Less than 0.05 mol or 0
．． If the amount is more than 2 moles, protamine mineral salt cannot be efficiently precipitated after cooling the hot water.

The hot water is cooled to below 10°C, preferably below 5°C. 1
If the temperature is higher than 0°C, protamine mineral salt cannot be efficiently precipitated.

There is no need to particularly adjust the pH of the hot water. When adjusting, add the protamine mineral salt and before adding the sulfate.
A range of 2 to 7 is desirable.

By performing purification according to the present invention, salts, acids, and contaminant proteins that adhere to the precipitate of protamine mineral salts when extracting and separating the milt can be removed.

According to the present invention, protamine mineral salts extracted and separated from fish milt can be efficiently purified by simple means such as recrystallization.

In the present invention, the solubility of protamine mineral salt is 0.05 to 0.05 to 0.05 to 0.05 to 0.05 to
This is based on the new finding that the concentration is minimized in a solution with an extremely low concentration of 0.2 mol.

Table 1 shows the relationship between its solubility (wt%) and sulfate concentration, taking procumin sulfate as an example in an aqueous solution with a water temperature of 5° C. and a pH of 2.5. In the table, the concentration represents the number of moles dissolved in solution lQ.

Table 1 From Table 1, the solubility of protamine sulfate is 0.05 at the salt concentration.
It can be seen that the amount becomes minimum in the range of ~0.2 mol.

The present invention will be explained in more detail using examples.

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

100g of ground salmon milt in 500ml of 5% (IN) sulfuric acid
1 and 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.

Mayuzumi 1 30 ml of concentrated ammonia water was added to the extract under stirring at room temperature 7+24°C, the pH was adjusted to 3, and 124 g of ammonium sulfate was added little by little to precipitate the contaminant proteins. The accumulated protein precipitate was removed by centrifugation to obtain 630 ml of supernatant.

Add 11 ammonium sulfate to the supernatant at room temperature of 24°C while stirring.
30 g was gradually added to salt out protamine sulfate. The salted out protamine sulfate is separated by centrifugation to obtain white candy-like protamine sulfate.

4g was obtained.

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 DEG C.) to give an ammonium sulfate concentration of 0.09 mol per liter of the solution. Thereafter, the solution was cooled to 5°C and left at that temperature for 2 days to precipitate protamine sulfate.

The supernatant liquid of precipitated protamine sulfate was removed by decantation to obtain protamine sulfate in the form of a transparent oil. It was freeze-dried to obtain 4.76 g of white protamine sulfate (yield: 4.76
%).

Example 2 Extraction process Frozen salmon milt was thawed, washed with water, drained, and ground.

IKg of ground salmon milt and 2.35 moles of ammonium sulfate were added to 2 liters of 10% (2N) sulfuric acid,
The mixture was stirred at 0°C for 1 hour. After stirring, the residue was removed by centrifugation to obtain 2.5 liters of protamine sulfate extract.

The extract contained 53.3 g (2.13% by weight) (dry equivalent) of protamine sulfate.

Separation Step 2.5 liters of the extract was cooled to 5° C. and allowed to stand at that temperature for 16 hours to separate the protamine sulfate precipitate and the supernatant. The supernatant liquid was collected by decantation.

The precipitate of protamine sulfate is white and candy-like, and the yield is 102g.
Met.

The amount of supernatant liquid collected was 2.43 liters.

Ingredients: 7.26% sulfuric acid, 1.72 mol/liter ammonium sulfate, 1.6% by weight of protein contaminants, 0 protamine sulfate.
．． It was 4% by weight (9.72g).

Purification process 102g of protamine sulfate obtained in the separation process was transferred to 800m
1 of hot water (40-50'C) to give a concentration of about 0.14 mole ammonium sulfate per liter of solution. Thereafter, the solution was cooled to 5° C. and allowed to stand overnight at that temperature to separate the protamine sulfate precipitate and the supernatant liquid.

The supernatant liquid was removed by decantation to obtain protamine sulfate as a clear oil. It is lyophilized to produce white protamine sulfate 43.
．． 2g was obtained (yield 4.32%).

Comparative Example 1 The same procedure as in Example 1 was carried out except that the plate-making process in Example 1 was not performed. The obtained white candy-like protamine sulfate was freeze-dried to obtain 6.30 g of white protamine sulfate powder.
(yield: 6.30%).

Comparative Example 2 The same procedure as in Example 2 was carried out except that the rib step in Example 2 was not performed. The obtained white candy-like protamine sulfate was freeze-dried to obtain 53.2 g of white protamine sulfate powder.
(Yield 5.32%) was obtained.

Example 3 The protamine sulfate obtained in Examples 1 and 2 and Comparative Example 1.2 was used as a sample, and its purity was measured.

Purity was examined by chromatography, absorption analysis, solubility of protamine sulfate in water, and antibacterial activity.

High performance liquid chromatography was used for chromatography. The purity of test 1:1 was compared using protamine sulfate (produced by Salmon, manufactured by Wako Pure Chemical Industries, Ltd.) as a standard, and the purity was determined as 1.00, and the purity was expressed as HPLC purity. Further, the product of the HPLC purity and the yield of the sample was shown as the standard conversion yield. Here, the standard conversion yield refers to the yield when the amount of protamine sulfate obtained from 100 g of milt is converted to protamine sulfate nails with purity equivalent to that of the standard product, and is considered to be a measure for comparing the separation efficiency of protamine sulfate. It's good. The results are shown in Table 2.

A comparison of the HPLC purity shows that the purity of protamine sulfate obtained according to the present invention is about 1.2 times better than that of the comparative example.

Antibacterial activity was measured using protamine sulfate (produced by salmon) manufactured by Wako Hyakuyaku Kogyo Co., Ltd. as a standard, and Bacillus subtilis (B.
The antibacterial activity against S. 5ubtilis I AM 1069 strain was set at 1.00, and the antibacterial activity was expressed as specific activity. In addition, the product of the antibacterial activity and the sample yield was shown as the standard conversion activity yield.

Active yield in terms of standard is the yield when converted to the amount of standard required to show the same antibacterial activity as the amount of protamine sulfate obtained from 100 g of milt. Comparison of separation efficiency of protamine sulfate It can be thought of as a measure of The results are shown in Table 2. In addition to comparing the antibacterial activity and specific activity, the sulfuric acid polypropylene C obtained according to the present invention! Tamin is about 1% higher than that of the comparative example.
．． It turns out it's twice as good.

Table 2 Absorption analysis measured the absorbance at 260 nm due to nucleic acids and at 280 nm due to contaminant proteins.

The measurement was carried out using an aqueous solution of the sample having a concentration of 0.1% by weight at a temperature of 20°C. The results are shown in Table 3. Pure protamine sulfate has no absorption for light above 220 ru++. As can be seen from Table 3, the protamine sulfate obtained according to the present invention was compared with the protamine sulfate obtained according to the comparative example.
It can be seen that the amount of contaminant protein is extremely small.

Table 3 Visual observation of the solution state is a 2% aqueous solution (20°C) of each sample.
I did it. Both the protamine sulfate obtained according to the present invention and the comparative example were completely dissolved, and no insoluble matter was observed.

Effects of the Invention According to the present invention, protamine mineral salt obtained from fish milt can be purified without loss and through a simple process.

Claims (1)

[Claims] 1. Protamine mineral salt obtained by extraction and separation from fish milt is dissolved in hot water, and then a small amount of sulfate is added to the hot water and cooled to precipitate the protamine mineral salt. A method for purifying protamine mineral salt.