JPS636049B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for concentrating kallidinogenase contained in human urine. More specifically, the present invention relates to a method for concentrating kallidinogenase in human urine to efficiently separate kallidinogenase from other biologically active substances.

Kallidinogenase is an enzyme that acts on kininogen in plasma to specifically release kinin, and is used in various tissues and body fluids, including the submandibular gland, sweat glands,
It is also known to be distributed in the salivary glands, liver, small intestine, serum, and urine. Its pharmacological actions include smooth muscle contraction, capillary permeability enhancement, vasodilation, blood pressure lowering, leukocyte aggregation, pain induction, and catecholamine secretion. Therefore, currently, kallidinogenase preparations are used in the internal medicine field for the treatment of cerebrovascular disorders, coronary heart disease, and hypertension, in the surgical field for the treatment of limb peripheral blood circulation disorders due to various causes, and in the ophthalmology field for the treatment of peripheral blood circulation disorders in the extremities due to various causes. It is a useful drug that is used for lowering blood pressure, etc., and is also widely used in the otorhinolaryngological field to treat Meniere's syndrome, neurological tinnitus, etc.

A large number of methods for purifying and recovering physiologically active substances present in human urine, including kallidinogenase, have been proposed. However, such conventional methods specifically purify and recover individual physiologically active substances from a mixture of various physiologically active substances, but they do not allow the interaction of various medically useful physiologically active substances present in human urine. An efficient separation method that takes into account the relationship between the two has not yet been established.

Therefore, in the conventional technology, in the recovery of various effective physiologically active substances from human urine, the operations are troublesome, the purity and recovery rate are reduced, and the preparations are relatively expensive. It was an unfavorable situation.

An object of the present invention is to provide an efficient industrial production method for physiologically active substances present in human urine. That is, the object of the present invention is to provide a method for effectively purifying and recovering kallidinogenase present in human urine, as well as facilitating the separation and recovery of other physiologically active substances.

According to the present invention, human urine and colloidal hydrated silicic acid are
Contact at pH 5.5-9 to adsorb most of the physiologically active substances other than kallidinogenase, contact the supernatant with an adsorbent containing silicon at pH 3.0-5.2 to adsorb kallidinogenase, and elute the adsorbed kallidinogenase. A method for concentrating kallidinogenase in human urine is provided.

The present inventors have discovered that colloidal hydrous aluminum silicate selectively adsorbs kallidinogenase from human urine and has arrived at the present invention. i.e. PH3.0
~5.2, approximately 30 to 70% of kallidinogenase in human urine is selectively adsorbed, whereas the majority of important physiologically active substances such as lysozyme are adsorbed when the pH is higher than the upper limit. It turned out that it was.

In the present invention, it is preferable that the human urine used is fresh and not putrefied. The pH of urine is 5.5
If the value is 9 to 9, it may be used as is, but it may be adjusted within that range, more preferably from 5.7 to 8.5. Further, it is preferable to remove precipitates or suspended matter in urine that may occur during pH adjustment by conventional methods such as centrifugation, filtration, dialysis, etc.

The colloidal hydrated aluminum silicate used in the present invention is a compound mainly composed of SiO ₂ and Al ₂ O ₃ and having a SiO ₂ /Al ₂ O ₃ ratio of 4.4 to 7.7 (average 5.9).
The amount of colloidal hydrous aluminum silicate to be used is sufficient to be 50 to 500 mg per 1 part of the raw urine, but there is no particular limitation if it is more than this, apart from the problem of yield.

After washing as a pretreatment, this colloidal hydrated aluminum silicate is added to the urine in the required amount, brought into contact with physiologically active substances contained in the urine, and adsorbs substances other than kallidinogenase, such as lysozyme. The contact time is about 30 minutes to 5 hours, and in the case of a batch method, stirring is more preferred. As a treatment method, a batch method is sufficient, but a column chromatography method may also be used. The treatment temperature is not particularly limited, and room temperature is sufficient, but more preferably
It is 30℃. After sufficient adsorption has taken place, the adsorbent and supernatant are separated. This separation may be performed by a gradient method or by centrifugation, and is not particularly limited.
After washing the separated colloidal hydrous aluminum silicate, the physiologically active substance adsorbed to the adsorbent can be eluted and recovered.

The supernatant remaining without being adsorbed to the adsorbent is
Used as a raw material for the production of kallidinogenase.

The supernatant is used as it is, preferably after centrifugation, and then the pH is adjusted to 3.0 to 5.2, preferably 4 to 5. In order to recover the contained kallidinogenase from this aqueous solution, the aqueous solution is brought into contact with an adsorbent containing silicon, and the kallidinogenase is adsorbed onto the adsorbent and recovered. As an adsorbent containing silicon,
Colloidal hydrated aluminum silicate, silica gel, magnesium aluminate silicate, silica glass, diatomaceous earth, and the like can be used. These adsorbents are washed, adjusted to pH 3 to 5.2, preferably equilibrated with a buffer having a pH in this range, and then contacted with the aqueous solution containing the kallidinogenase by a batch method or a column chromatography method. let The contact conditions are approximately 30 minutes to 5 hours.
Room temperature is sufficient, but preferably 5-30℃
It is. The amount of adsorbent added is 50 to 1000 mg per 1 aqueous solution containing kallidinogenase, and after adsorbing kallidinogenase, in the case of batch method, it is separated by tilting or centrifugation, and the adsorbent is collected, and in the case of column chromatography method, it is separated. After washing the adsorbent, kallidinogenase is eluted with an elution buffer.

Elution is an alkaline aqueous solution with a pH of 9 to 12, 0.3M.
The electrolyte solution containing the above salt concentration or an aqueous solution consisting of a combination thereof is used. For example,
0.15-0.2M phosphate buffer with PH10 containing 0.1M-0.5M NaCl, alkaline solution containing 0.5M-2M basic amino acids, ammonia water with a concentration of about 2% or more, 1M concentration or more with a pH of 10 or more saline solution of
Alternatively, an ammonium sulfate aqueous solution with a pH of 10 or higher and a concentration of 0.5M or more, a 5-10% neutral amino acid aqueous solution, etc. are used.

In the thus obtained aqueous solution, kallidinogenase is recovered in a concentrated state, and this product is further purified to a high degree according to a known method. For example, a method using a weakly basic anion exchange resin (JP
155608), a method using immobilized concanavalin A (Japanese Unexamined Patent Publication No. 52-18883), and a method combining two or more types of anion exchangers, gel filtration, etc. [J.
Biochem. 80 671 (1976)] etc. are used. A kallidinogenase preparation that can be used for medical purposes is obtained in accordance with the well-known conventional techniques for manufacturing pharmaceuticals. Note that heat treatment for virus inactivation can be performed in the purification step.

The method of the present invention, which consists of the above, not only makes it possible to efficiently recover kallidinogenase from human urine, but also facilitates the acquisition of other physiologically active substances, and its commercial value is extremely high. expensive.

Examples are given below to explain the present invention in more detail.

In the Examples, the method for measuring kallidinogenase activity (measuring method for increasing blood flow) was described by Moriya et al.
al) [H.Moriya, K.Yamazaki, and H.
Fukushima, J.Biochem., 58 , 201 (1965)]. That is, using an adult mongrel dog weighing approximately 10 kg, pentobarbital (25 to 50
Anesthetized by intraperitoneal injection of mg/Kg), the femoral artery was exposed, and heparin was injected intravenously (3000-5000 units).
Thereafter, a cannula was inserted into the femoral artery, and the blood was returned to the distal side of the femoral artery via an electromagnetic flowmeter (Nihon Kohden MF-2 model). Minute changes in blood flow captured by an electromagnetic flowmeter are transferred to a recorder (Nihon Kohden RJG-3022) via a DC amplifier.
was recorded.

The standard kallidinogenase includes porcine pancreatic kallidinogenase 751-M purified by the inventors.
(26.7KU/mg) was used. The activity was expressed in KU (kallidinogenase unit).

Example 1 After washing 50 g of colloidal hydrated aluminum silicate with a sufficient amount of distilled water, it was washed with 0.1 M phosphate buffer.
Equilibrate at PH6.0. Approximately 100 samples of fresh urine collected from an adult male are adjusted to pH 6.0 and brought into contact with colloidal hydrated aluminum silicate. After stirring and contacting for about 2 hours, the mixture is allowed to stand still and separated into a supernatant and an adsorbent.
Physiologically active substances adsorbed on the adsorbent are collected separately. Adjust the pH of the supernatant to 4.5 with 5N HCl,
Add 100% colloidal hydrated aluminum silicate to this.
g and contact with an aqueous solution containing kallidinogenase. After stirring for about 2 hours, the mixture is allowed to stand to separate the colloidal hydrated aluminum silicate. After washing with a sufficient amount of cold water, the kallidinogenase is eluted with a 10% W/V ammonium sulfate aqueous solution (PH adjusted to about 11 with NaOH). The eluate is brought to about 75% ammonium sulfate saturation and the precipitate is collected. After dissolving the precipitate in water, it was dialyzed against 0.1M phosphate buffer at pH 7.5.
Desalting was performed to obtain a concentrated solution.

Add Cephadex G-100 to the obtained concentrate,
After suspending and swelling in physiological saline, remove the solvent.
Substitute with 0.05M phosphate buffer (PH7.0), swell further, fill in a column (2.5 x 90cm), and perform gel filtration through a column equilibrated with the same buffer overnight to collect the kallidinogenase fraction. did.

QAE-Sephadex A-50 was washed several times alternately in a 0.5N aqueous thorium hydroxide solution, purified water, and a 0.5N hydrochloric acid solution, and finally washed with purified water until the washing solution became near neutral. Next, after equilibrating with 0.05M phosphate buffer (PH7.0), the column (2.5 x 90cm)
and equilibrated with the same buffer overnight. The kallidinogenase fraction was added to this column in two portions. Elution was performed with a concentration gradient of 0-0.5M saline solution. As a result, kallidinogenase
It eluted around 0.3M.

After concentrating this effective fraction, Sephadex G-
100 (a 2.5 x 90 cm column washed with a sufficient amount of water) was used for gel filtration and desalting, and the kallidinogenase active fraction was collected and freeze-dried to obtain the final purified sample. The specific activity of this preparation is
20KU/A280, and the recovery rate from urine was 50%.

Example 2 The active fraction of kallidinogenase after the QAE-Sephadex treatment in Example 1 was dialyzed against purified water overnight, and then a DEAE-cellulose column (5.0x) equilibrated with 0.05M phosphate buffer (PH7.0) 70cm)
was filled and adsorption was performed. Elution was performed using a concentration gradient of 0 to 0.5 M saline solution, and a fraction with kallidinogenase activity was collected. This product was dialyzed against purified water overnight, filtered for sterilization, and freeze-dried to obtain the final purified sample. The specific activity of this preparation is
It was 30KU/A280.

Example 3 Example 1 is repeated except that Columnite (Fuji Chemical Co., Ltd., magnesium aluminate silicate) is used instead of the colloidal hydrous aluminum silicate that is contacted with the supernatant in the kallidinogenase concentration of Example 1. Almost the same results were obtained.

Example 4 Example 2 was repeated with almost the same results, except that the active fraction of kallidinogenase after the QAE-Sephadex treatment in Example 2 was heated at 60°C for 10 hours prior to dialysis against purified water. I got it.

Claims (1)

[Claims] 1. Human urine and colloidal hydrous aluminum silicate are brought into contact at a pH of 5.5 to 9 to adsorb most of the physiologically active substances other than kallidinogenase, and the supernatant and an adsorbent containing silicon are brought into contact at a pH of 3.0 to 5.2. 1. A method for concentrating kallidinogenase in human urine, which comprises adsorbing kallidinogenase through contact and eluting the adsorbed kallidinogenase.