JPS5949239B2 - Simple purification method for high-purity protamine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a simple method for purifying high purity protamine.

Protamine is a strong basic protein with a relatively small molecular weight and high arginine content that 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 long-acting insulin preparations, which are complexes with insulin, and protamine injections, which are anti-heparin 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 extracting protamine from sperm nuclei of mature milt of the fish with dilute mineral acid, or by extracting nucleoprotamine with concentrated saline and then separating protamine.

However, since these extracts contain proteins other than protamine, peptides, amino acids, nucleic acid degradation products, lipids, and their degradation products, protamine isolated using an appropriate precipitant can be used. Mineral salts require considerable purification. Conventional purification methods for protamine include (1) repeated reprecipitation with alcohol as sulfate, (2) purification through treatment with picrate, or (3) other general protein precipitating agents ( platinum chloride,
(tungstic acid, metaphosphoric acid, ferrocyanic acid, sulfosalicylic acid, etc.) or (4) separation and purification using ion exchange resin. In addition, when extracting as nucleoprotamine, (1) salt concentration (2) Separate and purify brotamine by salting out from a salt solution of nucleoprotamine after reprecipitation purification by lowering the nucleoprotamine, or (3) Separate and purify the nucleic acid copper salt with copper sulfate or copper chloride. Various methods have been used to obtain protamine mineral salts by removing them as protamine mineral salts. In any case, these conventional purification methods are either unsuitable for mass production due to complicated operations or require expensive materials, or are unsuitable for purification for clinical drug use. However, if the desired purity is desired, the yield of brotamine will be significantly reduced, which is economically disadvantageous.

The feature of the present invention is that the protamine mineral salt containing protamine is extracted and separated from the milt containing protamine by a conventional method, and the impurity-containing protamine mineral salt is treated with an adsorbent in an acidic dilute aqueous solution. By performing fractional precipitation, it is possible to extremely easily purify high-purity protamine, which can be sufficiently applied as a clinical drug.

The relative amounts of impurities such as proteins other than protamine and their decomposition products, nucleic acid decomposition products, lipids and their decomposition products, etc. are not necessarily uniform depending on the raw material Milt, extraction conditions, separation conditions, etc. Based on the principle of removing impurities, it is practically difficult to set targets for each impurity and perform optimal removal and purification treatment. Requires.

In an acidic dilute aqueous solution of protamine mineral salt, the chemical bonds and physical bonds between the above impurities and protamine are almost completely broken or dissolved in a separated state, and substances other than protamine are significantly attached to the adsorbent. Based on the new knowledge that there is an affinity for protamine, they established the present invention, which allows extremely simple purification of high-purity protamine.

Of course, it is of a non-adsorbable nature and naturally remains in the solution, so it is important that the amount of organic solvent used in the precipitation operation is the minimum necessary amount.

The more dilute the concentration of the protamine aqueous solution, the better.
It can be freely selected within the range of 1 to 10%. The stronger the acidity, the better the solubility of impurities, and it is usually desirable that the acidity be 3 or less. Any general adsorbent (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 10q6. The temperature conditions may be hot or cold, and the longer the treatment time, the better the results. The organic solvent may be any alcohol, acetone, etc., and the amount used is preferably at most the equivalent volume. Examples and reference examples are given below.

Reference Example 0.5N hydrochloric acid 601 was added to 25 kg of mature herring milt, and the mixture was extracted at room temperature for 5 hours while circulating the liquid.

The separated extract was adjusted to Pl{3 to 4 with aqueous ammonia, and an aqueous solution of sodium metaphosphate 6009 was added thereto with stirring and left overnight. The supernatant liquid was decanted, and candy-like protamine metaphosphate 1.9k9 was dissolved in 41 2M ammonium sulfate under heating and left overnight. The lower layer of oily protamine sulfate is dehydrated and solidified with alcohol, dried and
．． 0 kg of protamine sulfate white powder is obtained. Example: Protamine sulfate 1k9 prepared in the reference example was dissolved in 201 of water by heating, and adjusted to 1.5 with dilute sulfuric acid.

After adding 50 g of activated carbon and stirring for 1 hour, the mixture was separated.

511 methanol is added to a colorless and transparent aqueous protamine sulfate solution while stirring, and the mixture is left overnight. Decant the supernatant liquid and add 5 drops of candy-like protamine sulfate! Dehydrate and solidify with methanol. A pure white crystalline product 8509 of high purity protamine sulfate is obtained. 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 Purification of high-grade protamine characterized by treating an acidic dilute aqueous solution of protamine mineral salt obtained by extraction and separation from fish milt containing protamine with an adsorbent, and then precipitating it fractionally with the minimum necessary amount of an organic solvent. Law.