JPH0220235B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for adding and adsorbing human urine with an adsorbent, and extracting human urine kallikrein (hereinafter abbreviated as HUKN) from the adsorbent with good yield and high purity. Kallikrein is a proteolytic enzyme produced in various parts of an animal's body that breaks down kininogen in serum to produce kinin, which has long been known to have effects such as lowering blood pressure and increasing blood flow. ing. Methods for concentrating HUKN include a method using silica gel (Japanese Patent Publication No. 46-19067), a method using arginine-cepharose (Japanese Patent Publication No. 55-99191), and a highly cross-linked macroporous anion using a tertiary amine as an exchange group. Method using exchange resin
5515) etc. have been reported. However, these methods have high purity, recovery rate, cost,
It cannot be said that this method is necessarily satisfactory in terms of ease of operation. As a result of further research, the present inventors found that hot extraction from kaolin or chitosan adsorbed with HUKN is more efficient and effective than cold extraction, as shown in the experiment below. We discovered that it was possible to extract highly pure HUKN. (Experiment 1) After adjusting the pH of healthy male urine to 4.0, it was divided into 1 portion and 10 g of kaolin was added to each portion to adsorb HUKN.
An adsorbent was obtained. Next, after washing the adsorbent, each pH5
-11 buffer solution at room temperature for 1 hour and 12 hours, and then at 60°C for 1 hour and 12 hours, a HUKN extract was obtained, and the results shown in Table 1 were obtained.

[Table] (Experiment 2) The same experiment as above was conducted except that the urine was adjusted to pH 5.0 and chitosan was used instead of kaolin, and the results are shown in Table 2.

[Table] As is clear from the above results, when extracting from kaolin or chitosan adsorbed with HUKN, the method of extraction at 60°C for 12 hours is better than the method of extraction at room temperature for 1 hour, which results in a pH within the range of 7-10. We were able to obtain HUKN with a higher degree of purification. (Experiment 3) After adjusting 100 of healthy male urine to PH4.0, 50
The mixture was divided into two parts, 0.5 kg of kaolin was added to each part, stirred for 1 hour to adsorb HUKN, and filtered to obtain a HUKN adsorbent. Next, the adsorbent was thoroughly washed with pure water. and each to its adsorbent
Add 0.1M-Tris-HCl buffer, PH9.0, extract at room temperature for 1 hour, and heat the other at 60°C for 12 hours, filter and filtrate.
1 of each extract was obtained. The total activity of HUKN in the former is 3780 units (recovery rate 42
%), and the purity was 11 units/mg of protein. The total activity of the latter HUKN was 6660 units (recovery rate 74
%), and the purity was 29 units/mg of protein. Next, each extract was dialyzed against 0.05M phosphate buffer containing 0.05M sodium chloride, pH 7.5.
It was adsorbed onto a DEAE-cellulose column (diameter 3 cm x height 20 cm) buffered with the same buffer, and after washing with the same buffer containing 0.5 M sodium chloride.
HUKN was obtained by elution with 0.05M phosphate buffer, pH 7.5. The total activity of HUKN in the former is 3060 units (recovery rate 34
%), and the purity was 85 units/mg of protein. The total activity of the latter HUKN was 5760 units (recovery rate 64
%), and the purity was 240 units/mg of protein. (Experiment 4) When the same experiment as (3) was conducted except that the urine was adjusted to pH 5.5 and chitosan was used instead of kaolin, the total activity of HUKN in the room temperature extract was 6120 units (recovery rate 72%). ), purity is 32 units per mg of protein,
The total activity of HUKN in the heated extract was 7820 units (recovery rate 92%), and the purity was 74 units per mg of protein.
Next, each extract was dialyzed against 0.05M phosphate buffer containing 0.05M sodium chloride, pH 7.5, and adsorbed onto a DEAE-cellulose column (3 cm in diameter x 20 cm in height) buffered with the same buffer. , after washing with the same buffer containing 0.5M sodium chloride.
HUKN was obtained by elution with 0.05M phosphate buffer, pH 7.5. In addition, after purification using a DEAE-cellulose column, the total activity of the former HUKN was 5015 units (recovery rate 59%), and the purity was 1 mg protein.
The total activity of the latter HUKN was 7140 units (recovery rate 84%), and the purity was 510 units per mg of protein. Based on the above results, HUKN was extracted from kaolin or chitosan with hot water.
Since heat-sensitive contaminant proteins and proteases have been removed from HUKN, the recovery rate of subsequent purification is lower than that of HUKN extracted with cold water.
It had excellent purity. The present invention is based on these new findings, and involves adding a neutral or basic buffer to kaolin or chitosan on which human urine kallikrein has been adsorbed, and heating the mixture at 50-70°C.
This is a method for extracting human urine kallikrein, which is characterized by heating it to . Examples of the buffer include phosphate buffer, Tris buffer, and ammonia buffer. Any buffer solution may be used as long as it is neutral or basic, that is, has a pH of 7.0 or higher, but the preferred pH is 7 to 10.
and more preferably 8-10. The above buffer solution is added to the adsorbent adsorbed with HUKN, and the HUKN is extracted by heating it to 50 to 70°C. If the extraction temperature is lower than 50℃, both extraction efficiency and specific activity will decrease, and if it exceeds 70℃, HUKN
tends to become inactive. The extraction time depends on the type of adsorbent, but is usually 8.
~24 hours, preferably 10-18 hours. Example 1 Healthy adult male urine 10 was adjusted to pH 3.5, 100 g of kaolin was added, stirred for 1 hour to adsorb HUKN, and filtered to obtain a HUKN-adsorbent.
Next, the adsorbent was thoroughly washed with pure water. Then, from the adsorbent, 0.2M-Tris-HCl buffer, PH10.0
was added, heated to 65°C, extracted for 10 hours, and filtered to obtain 300 ml of HUKN extract. obtained
The total activity of HUKN was 1599 units (recovery rate 82%), and the purity was 32 units per mg of protein. Example 2 10 samples of urine from a healthy adult male were adjusted to pH 3.5, 100 g of kaolin was added thereto, and kallikrein was adsorbed by stirring for 1 hour. A filtered HUKN-adsorbent was obtained. Furthermore, the adsorbent was thoroughly washed with pure water. Then, 0.2M Tris-HCl buffer, pH 9.0 was added to the adsorbent, heated to 65°C, extracted for 10 hours, and filtered to obtain 300 ml of HUKN extract. The total activity of the obtained HUKN was 1388 units (recovery rate 75%), and the purity was 31 units per mg of protein. Example 3 100 g of healthy adult male urine was adjusted to pH 3.5, 100 g of kaolin was added, and the mixture was stirred for 1 hour to adsorb HUKN, followed by filtration to obtain a HUKN-adsorbent.
Furthermore, the adsorbent was thoroughly washed with pure water. Next, add 0.2M Tris-HCl buffer to the adsorbent, pH 9.5.
was added, heated to 70°C, extracted for 8 hours, and filtered to obtain 300 ml of HUKN extract. obtained
The total activity of HUKN was 1311 units (recovery rate 69%), and the purity was 40 units per mg of protein. Example 4 Healthy adult male urine 10 was added with 2N-hydrochloric acid to make the pH 5.0.
After adding 100 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.0, was added to the adsorbent, heated to 60°C, extracted for 12 hours, and filtered to obtain 300 ml of HUKN extract. The total activity of HUKN obtained was 1886 units (recovery rate
(92%) purity was 73 units/mg protein. Example 5 Healthy adult male urine 10 was added with 2N-hydrochloric acid to make the pH 5.5.
After adding 100 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.0, was added to the adsorbent, heated to 50°C, extracted for 18 hours, and then filtered.
300 ml of HUKN extract was obtained. The total activity of HUKN obtained was 1655 units (recovery rate
93%), and the purity was 68 units/mg of protein. Example 6 Healthy adult male urine 10 was added with 2N hydrochloric acid to make the pH 5.5.
After adding 100 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.0, was added to the adsorbent, heated to 70°C, extracted for 10 hours, and then filtered.
300 ml of HUKN extract was obtained. The total activity of HUKN obtained was 1330 units (recovery rate
70%), and the purity was 75 units/mg of protein. Example 7 Healthy adult male urine 20 was added with 2N-hydrochloric acid to make the pH 4.0.
After adding 150 g of kaolin and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.5, was added to the adsorbent, heated to 60°C, extracted for 12 hours, and filtered to obtain 600 ml of HUKN extract. The total activity of HUKN obtained was 2774 units (recovery rate
73%), and the purity was 32 units/mg of protein.
Next, the extract was dialyzed against 0.1M phosphate buffer, pH 7.0, and adsorbed onto a DEAE-cellulose column (diameter 3 cm x height 20 cm) buffered with the same buffer. After washing, it was eluted with 0.1M phosphate buffer containing 0.4M sodium chloride, pH 7.0 to obtain a HUKN solution. The total activity of HUKN obtained was 2356 units (recovery rate
62%), and the purity was 235 units/mg of protein. Example 8 Healthy human urine Male urine 20 with 2N-hydrochloric acid added to pH 4.0
After adding 150 g of kaolin and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.5, was added to the adsorbent, heated to 60°C, extracted for 12 hours, and filtered to obtain 600 ml of HUKN extract. Obtained HUKN
The total activity was 2664 units (recovery rate 72%), and the purity was 35 units per mg of protein. Next, the extract
A precipitate formed by adding 300 g of ammonium sulfate is obtained by centrifugation. This precipitate was dissolved in 20 ml of 0.2 M Tris-HCl buffer, pH 8.5, and a Sephadex G-100 column (diameter 2.6 cm) was buffered with the same buffer.
x height 100cm)
110 ml of HUKN was obtained. The total activity of KUKN obtained was 2405 units (recovery rate
65%), and the purity was 220 units/mg of protein. Example 9 Healthy adult male urine 20 was added with 2N hydrochloric acid to pH 3.5
After adding 150 g of kaolin and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.5, was added to the adsorbent, heated to 66°C, extracted for 12 hours, and filtered to obtain 600 ml of HUKN extract. Obtained HUKN
The total activity was 2880 units (recovery rate 70%), and the purity was 35 units per mg of protein. Next, the extract
Dialyzed against 0.1M sodium bicarbonate buffer containing 0.5M sodium chloride, pH 9.0, and adsorbed onto an aprotinin-Sepharose column (diameter 2.0cm x height 20cm) buffered with the same buffer. - 20 ml of HUKN solution was obtained by elution with 0.1M acetate buffer containing sodium chloride, pH 3.5. The total activity of HUKN obtained was 1700 units (recovery rate
50%), and the purity was 480 units/mg of protein. Example 10 Healthy adult male urine 20 was added with 2N-hydrochloric acid to make the pH 5.0.
After adding 200 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, add 0.2M Tris-HCl buffer, pH 9.0, to the adsorbent.
After heating to 60℃ and extracting for 12 hours, filtration was performed.
750 ml of HUKN extract was obtained. The total activity of the HUKN obtained was 3276 units (recovery rate 91%), and the purity was 73 units per mg of protein. Next, the extract
Dialyzed against 0.1M phosphate buffer, pH 7.0, adsorbed onto a DEAE-cellulose column (diameter 3cm x height 20cm) buffered with the same buffer, washed with the same buffer, and then 0.4M- Contains sodium chloride
A HUKN solution was obtained by elution with 0.1M phosphate buffer PH7.0. The total activity of HUKN obtained was 2952 units (recovery rate
82%), and the purity was 495 units/mg of protein. Example 11 Healthy adult male urine 20 was added with 2N-hydrochloric acid to make the pH 5.0.
After adding 200 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.5, was added to the adsorbent, heated to 60°C, extracted for 12 hours, and filtered to obtain 500 ml of HUKN extract. Obtained HUKN
The total activity was 3588 units (recovery rate 92%), and the purity was 76 units per mg of protein. Next, the extract
A precipitate formed by adding 250 g of ammonium sulfate is obtained by centrifugation. This precipitate was dissolved in 20 ml of 0.2 M Tris-HCl buffer, pH 8.5, and passed through Sephadex G-100 (diameter 2.6 cm x height 100 cm) buffered with the same buffer to obtain 105 ml of HUKN.
I got it. The total activity of KUKN obtained was 3315 units (recovery rate
85%), and the purity was 465 units/mg of protein. Example 12 Healthy adult male urine 20 was added with 2N-hydrochloric acid to make the pH 5.0.
After adding 200 g of chitosan and stirring for 1 hour, filtration was performed to obtain a HUKN-adsorbent. Furthermore, the adsorbent was thoroughly washed with pure water. Next, 0.2M Tris-HCl buffer, pH 9.0, was added to the adsorbent, heated to 60°C, extracted for 12 hours, and filtered to obtain 600 ml of HUKN extract. Obtained HUKN
The total activity was 3150 units (recovery rate 90%), and the purity was 72 units per mg of protein. Next, the extract
Dialyzed against 0.1M sodium bicarbonate buffer containing 0.5M sodium chloride, pH 9.0, and adsorbed onto an abrotinin-Sepharose column (diameter 2.0cm x height 20cm) buffered with the same buffer. M-
0.1M acetate buffer containing sodium chloride, PH3.5
Elution was performed to obtain 20 ml of HUKN solution. The total activity of HUKN obtained was 1820 units (recovery rate
52%), and the purity was 1050 units/mg protein. In addition, the unit of HUKN in Examples 1 to 12 is
Vasodilation measurement method (Journal of Biochemistry,
58, 201, 1965) and used it to perform the fluorescent synthetic substrate method (Journal of
Biochemistry, 82 , 1495, 1977). Protein content was measured using the Lowry-Folin method using bovine serum albumin as a standard.

Claims (1)

[Claims] 1. A method for extracting human urine kallikrein, which comprises adding a neutral or basic buffer to kaolin or chitosan on which human urine kallikrein has been adsorbed, and heating the mixture to 50-70°C.