JPH0141310B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for recovering kallikrein contained in human urine with good yield and high purity.

Kallikrein is an enzyme that acts on kininogen in plasma to specifically release kinin, and is distributed in various tissues and body fluids, including the submandibular gland, sweat glands, salivary glands, liver, small intestine, serum, and urine. Are known. Its pharmacological actions include smooth muscle contraction, capillary permeability enhancement, vasodilation, blood pressure lowering, leukocyte aggregation, pain induction, and catecholamine secretion. Kallikrein preparations are useful medicines that are widely used in various fields.In the field of internal medicine, they are used to treat cerebrovascular disorders, coronary heart disease, hypertension, etc.
In the surgical field, it is used to treat limb peripheral blood circulation disorders due to various causes, in the ophthalmology field, it is used to dilate peripheral retinal blood vessels and lower blood pressure in the eye fundus, and in the otolaryngology field, it is used to treat Meniere's syndrome, neurogenic tinnitus, etc. has been done.

A large number of methods have been proposed for the recovery of kallikrein, but the conventional methods require complicated operations and are not always sufficient in terms of yield and purity. For this reason, kallikrein preparations from human urine are relatively expensive, and conventional methods have not been satisfactory as industrial methods for producing kallikrein preparations.

The present invention aims to provide an industrial production method for recovering kallikrein present in human urine with good yield and high purity.

The present invention involves contacting human urine with colloidal hydrated aluminum silicate at pH 4.0 to 6.0, adsorbing most of the physiologically active substances other than kallikrein to the colloidal hydrated aluminum silicate, and separating the supernatant.
This supernatant is contacted with a water-insoluble trypsin inhibitor derived from animal organs equilibrated to pH 7.5 to 9.5 to adsorb kallikrein, and the adsorbed kallikrein is eluted from the trypsin inhibitor.

The raw material of the present invention is fresh human urine that has not putrefied.Human urine may be used as is if its pH is 5.5 to 9, but it is preferable to adjust the pH to 4.0 to 6.0. . In addition, suspended matter in urine and precipitates generated during pH adjustment are removed by conventional treatments such as centrifugation, filtration, and dialysis. The colloidal hydrated aluminum silicate used in the present invention has the ability to selectively adsorb physiologically active substances other than kallikrein from human urine, and at pH 4.0 to 6.0, the amount of physiologically active substances other than kallikrein in human urine is approximately 94%. ~100% is adsorbed. This colloidal hydrated aluminum silicate mainly consists of SiO ₂ and Al ₂ O ₃ , and the ratio of SiO ₂ /Al ₂ O ₃ is 4.4.
~7.7 (average 5.9). It is sufficient to use an amount of 50 to 500 mg per 1 part of the raw material urine, but a larger amount may be used, apart from the problem of yield.

After thoroughly washing colloidal hydrated aluminum silicate as a pretreatment, the required amount is added to human urine and brought into contact with physiologically active substances contained in the urine. adsorbs degrading enzymes). The contact time is about 30 minutes to 5 hours, and the treatment method may be a column chromatography method, but a batch method is sufficient, and in the case of the batch method, stirring is required. The treatment temperature may be room temperature, but is more preferably 5 to 30°C. After sufficient adsorption, the adsorbent and supernatant are separated by gradient method or centrifugation. The separated colloidal hydrated aluminum silicate is washed, and the adsorbed physiologically active substances are eluted and reused.

The supernatant after separating the adsorbent is used as a raw material for kallikrein. This supernatant can be used as it is or after centrifugation, and the pH should be adjusted to 7.5-9.5, preferably 8-8.
Adjust to 9. A water-insoluble trypsin inhibitor derived from animal organs is added to this supernatant to adsorb kallikrein. The animal organ-derived water-insoluble trypsin inhibitor is a known animal organ-derived trypsin inhibitor immobilized by a conventional water-insolubilization method. Kunitz's trypsin inhibitor [J.Gen.
Physiol., 19 991 (1936)], for example aprotinin. This trypsin inhibitor is washed and
After adjusting the pH to 7.5 to 9.5 and equilibrating with a buffer solution preferably having a pH in this range, the supernatant containing kallikrein is brought into contact with the supernatant containing kallikrein by a batch method or a column chromatography method. The contact time may be about 30 minutes to 20 hours, and the temperature may be room temperature, but preferably 5 to 30°C. The amount used is approximately 0.5 to 10 ml per 10 parts of the supernatant containing kallikrein, and after adsorbing kallikrein, the adsorbent is collected by gradient method or filtration in the case of batch method, and the column is washed in case of column chromatography method. Thereafter, kallikrein is eluted from the adsorbent using an elution buffer. Elution is preferably carried out with an acidic aqueous solution having a pH of 3 to 5, such as a 0.01 to 0.2M acetate buffer, a citrate buffer, a succinate buffer, or the like having a pH of 4.
The eluate thus obtained contains a large amount of highly pure kallikrein, and this eluate is purified to recover the desired kallikrein. Note that heat treatment is performed in the purification process to inactivate the virus.

The present invention can recover kallikrein contained in human urine with good yield and high purity, and is a satisfactory method for industrially producing kallikrein derived from human urine, and has extremely high value in the medical field.

The recovery rate of kallikrein from human urine according to the present invention is 45-75%, and the specific activity of the obtained kallikrein is 660-730 KU/A280 [unit: Frey.EK,
Kraut, H., and Werle, E., 1950,
“Kallikrein, Padutin”, Enke, Verlag,
Stuttgart], and its purity is determined to be 90-100% by disk electrophoresis on polyacrylamide gels [Davis,
O., 1964, Ann.NYAcad.Sci., 121, Art.2,
305].

The kallikrein activity was measured using HD-Val.
-L-Leu-L-Arg-P-nitroanilide
(Testzyme S-2266, Kabi Diagnostica
Sweden) was used as a substrate. This substrate is known to be highly specific for kallikrein derived from human urine. However, because it is also degraded by physiologically active substances other than kallikrein in urine, it cannot be said that only kallikrein can be accurately quantified. Therefore, SBTI (Soybean trypsin) was added to the reaction system.
Inhibitor) and aprotinin were added to serve as controls. In other words, kallikrein is
It is not inhibited by SBTI but is inhibited by aprotinin. On the other hand, urokinase, which is known to exist in human urine, is not inhibited by both SBTI and aprotinin, and many proteolytic enzymes such as trypsin are inhibited by SBTI. In addition to using a substrate with high specificity for kallikrein, kallikrein was accurately quantified by using measurement conditions that specifically quantify kallikrein.

Next, the present invention will be specifically explained with reference to Examples.

Example 1 Approximately 150 fresh urine collected from an adult male was adjusted to pH 5.0 and brought into contact with colloidal hydrated aluminum silicate (dry weight 50 g) washed with distilled water. After stirring at room temperature for about 1 hour to ensure sufficient contact, the supernatant is collected by centrifugation. Add and dissolve 787 g of tris(hydroxymethyl)aminomethane to supernatant 130, and adjust the pH to 8.5 with 3N-HCl. Next, add 50 ml of aprotinin/sepharose 4B equilibrated to PH 8.5 with 0.05 M Tris-HCl buffer.
The adsorption reaction was carried out by stirring at room temperature for about 20 hours. After filtering the reaction solution and filling the gel into a column, the PH
Wash the column with 0.05M Tris-HCl buffer for 8.5
Kallikrein was eluted with 0.05M acetate buffer PH4.0. In this way, kallikrein derived from human urine with a purity of 90% was recovered with a yield of 70%, and its specific activity was 700 KU/A280.

Example 2 After performing the same colloidal hydrous aluminum silicate treatment as in Example 1, the effective fraction of kallikrein was adjusted to pH 7.5, and this was concentrated 100 times using a hollow fiber eye with an ultramolecular weight of 10,000. 60℃,
Heat treatment was performed for 10 hours. Next, add and dissolve 7.9 g of tris(hydroxyamino)methylmethane,
Thereafter, the same aprotinin/cephalose treatment as in Example 1 was performed. The yield of kallikrein obtained is
70%, and the specific activity was 700KU/A280.

Claims (1)

[Claims] 1 Human urine and colloidal hydrated aluminum silicate
Most of the physiologically active substances other than kallikrein are adsorbed to colloidal hydrated aluminum silicate at pH 4.0 to 6.0, and the supernatant is separated. A method for recovering kallikrein from human urine, which comprises contacting a water-insoluble trypsin inhibitor to adsorb kallikrein and eluting the adsorbed kallikrein from the trypsin inhibitor.