JPS6230754B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an absorbent for enzyme purification, and more particularly to an adsorbent for affinity chromatography for purifying kallikrein with high purity and high yield. Kallikrein is an enzyme that acts on kininogen in serum to specifically release kinin. It is widely used clinically as a blood pressure lowering agent and a treatment for circulatory disorders in various organs because it exhibits smooth muscle contraction, blood pressure lowering, and blood flow increasing effects. Kallikrein has been purified using methods such as ammonium sulfate fractionation, acetone fractionation, gel filtration, and ion-exchange chromatography, but these methods require troublesome operations and are expensive. Purification to purity requires considerable effort and at the same time there are drawbacks in terms of yield. In particular, it is nearly impossible to purify kallikrein derived from human urine to a purity that can be intravenously injected into the human body using these methods. In recent years, affinity chromatography has become the preferred method for isolating enzymes. It is also known that carriers to which aprotinin is bound as a ligand via a spacer can be used for this purpose. For example, Cephalose 4B
Example of covalently bonding aprotinin to this activated carrier after activation with cyanogen bromide [Biochemical Pharmacology, 25 , 1607]
(1976)] and a carrier containing an activated ester of the carboxyl group of cephalose 4B-aminocaproic acid (manufactured by Pharmacia Fine Chemicals).
An example of covalently bonding aprotinin to Activated-CH-Sepherose 4B (this swelling carrier is activated to bind glycylleucine at a rate of 5 to 7 μmol per ml) [J.Immumology,
121 , 66 (1978)] are well known. As a result of additional trials of these methods, the inventors found that
For example, J.Immumology, 121 , 66 (1978). According to the method described in , the amount of aprotinin bound per ml of the swollen carrier is 1.2 μmol, and when kallikrein is purified using this carrier, the adsorption amount of kallikrein is extremely high, 1 to 5 nmol/ml of the swollen carrier. The adsorption molar ratio of kallikrein to immobilized aprotinin was less than 1%. The present inventors conducted various studies aimed at increasing the adsorption amount of kallikrein to a water-insoluble carrier containing 6-aminocaproic acid as a spacer and aprotinin as a ligand, and as a result, they discovered an extremely unexpected fact. In other words, the amount of kallikrein adsorbed onto the carrier is not simply affected by the amount of aprotinin bound as a ligand, but is significantly influenced by the degree of activation of the spacer bound to the carrier before binding aprotinin. By binding aprotinin while suppressing the activation level (the amount of glycylleucine bound per ml of swelling carrier) to about 1/2 or less than that of conventional products, more specifically to 1 to 3.5 μmol, aprotinin The present invention was completed by discovering that it is possible to produce an adsorbent for enzyme purification whose adsorption performance as a ligand is not impaired. The invention is ``6 as a spacer in a water-insoluble carrier.
- Aminocaproic acid is bound, its terminal carboxyl group is reacted with N-hydroxysuccinimide to form an active ester at a rate of 1 to 3.5 μmol per ml of the swollen carrier, and aprotinin is covalently bound to this adsorption for purification of kallikrein. It is about the body. Although the adsorbent for purifying kallikrein of the present invention does not particularly limit the amount of aprotinin bound,
Usually, it is appropriate to bind aprotinin at a rate of 0.024 to 0.31 μmol per ml of the swollen carrier, and the adsorbent thus prepared can adsorb 10 to 15 nmol of kallikrein per ml of the swollen carrier. If the adsorption amount of kallikrein is expressed as an adsorption molar ratio to immobilized aprotinin, it is approximately 3.5
This corresponds to ~47%, which is significantly higher than the 0.7~0.8% for the conventional method.Even when comparing the adsorption amount of kallikrein per ml of the swollen carrier, the 10~15nmol of kallikrein of the present invention is higher than that of the conventional method. This far exceeds the 1 to 5 nmol in the case of . The adsorbent of the present invention can be easily produced by appropriately combining reactions known per se. That is, in order to bind 6-aminocaproic acid to a water-insoluble carrier, conventional methods such as CDI (N·N'-carbonyldiimidazole) activation method and cyanogen bromide activation method can be used. Also,
To attach the ligand to the end of the spacer,
A carrier activated with a common condensing agent such as DCC [N·N'-dicyclohexylcarbodiimide] or EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and N-hydroxysuccinimide is prepared, and this Add aprotinin to bind. Any water-insoluble carrier may be used as long as it can bind 6-aminocaproic acid, and examples thereof include agarose, crosslinked dextran, celluloses, polysaccharides such as agar, and polymers such as polyacrylamide. In purifying kallikrein using the adsorbent of the present invention, the adsorbent is packed in a column, a crude kallikrein-containing liquid is passed through the column to adsorb kallikrein, and the column is filled with an alkaline aqueous solution with a pH of 7 or higher. After washing with water and eluting with an acidic salt solution, a single, high-purity kallikrein is electrophoretically produced at 90%
% or more in yield. Crude kallikrein was obtained from human urine using the usual method and had a specific activity of 0.005 to 0.035 U/A ₂₈₀ .
This is dialyzed against a 0.05M phosphate buffer (PH7.0-9.5) containing 0.2-0.6M salt, and then adsorbed through the column.
The same buffer as the crude kallikrein solution to be passed through the column is used as the washing solution. The salt concentration in the buffer is 0.2~
0.6M, preferably 0.3-0.5M. Kallikrein eluate has a salt concentration of 0.3 to 1.0M, preferably 0.5
A 0.1M acetate buffer (PH 4-5) containing ~1.0M is preferred. Next, the effects of the present invention will be illustrated with test examples. Test example Sepharose CL-4B (manufactured by Pharmacia Fire Chemicals) was activated, and 10 μmol of 6-aminocaproic acid was bound per ml of carrier.
Using EDC and N-hydroxysuccinimide, 6 ml of carriers with different degrees of activation were prepared.
Combine 3ml of this with glycylleucine,
After hydrolyzing this with 6NHCl at 110℃, 1 ml of glycylleucine swollen carrier was determined by amino acid analysis.
The amount of binding per unit was determined. Meanwhile, the remaining 3 ml was combined with aprotinin to create an adsorbent. The amount of bound aprotinin was determined by back calculation from the amount of unreacted aprotinin at this time. 3 ml of this adsorbent was packed into a column and loaded with an excess amount of crude kallikrein (0.035 U/A ₂₈₀ ). Next contains 0.5M salt
After washing with 0.05M phosphate buffer (PH8.5), it was eluted with 0.1M acetate buffer (PH4.0) containing 1M sodium chloride. The amount of kallikrein adsorbed per ml of carrier was determined by back calculation from the amount of unadsorbed kallikrein. The specific activity and recovery rate of kallikrein, which was eluted at the same time, were determined. The results of this test are shown in Table 1.

[Table] As is clear from the test results in Table 1, as the amount of glycylleucine bound increases, that is, the degree of activation of the carrier increases, the amount of aprotinin bound increases. However, as shown in No. 1, only 0.72% of the immobilized aprotinin bound to kallikrein. Further, as shown in No. 2, even when the amount of immobilized aprotinin was reduced to 50 nmol, the ratio of kallikrein to the amount of immobilized aprotinin remained almost the same. On the other hand, as the amount of glycylleucine binding decreases, that is, the degree of activation of the soybean decreases,
The amount of aprotinin bound decreases. However, the ratio of the amount of adsorbed kallikrein to the amount of immobilized aprotinin increased. The amount of glycylleucine bound is
When the activation degree of Gel was 1.8 μmol/ml, the adsorption amount of kallikrein was maximum. Furthermore, as shown in No. 1, when the amount of aprotinin per ml of carrier was large, the specific activity of the obtained kallikrein was low. In addition, as shown in No. 8, the spacer content is
When Gel was prepared at 2.3 μmol/ml and activated by the method described above, the amount of glycylleucine bound was 1.9 μmol/ml of carrier. When aprotinin was bound to this activated carrier, almost the same results as No. 5 were obtained. This indicates that when preparing the adsorbent of the present invention, it is sufficient to specify the degree of activation of the carrier rather than the spacer content per ml of the swollen carrier. From the above results, if an adsorbent in which aprotinin is bound to an activated carrier in which the amount of glycylleucine bound is in the range of 1 to 3.5 μmol per ml of carrier is used, kallikrein with high specific activity can be produced in good yield. can get. In the present invention, the activity of kallikrein is determined by the synthetic substrate HD-valyl-leucine-arginylparanitroanilide (manufactured by Kabi, Sweden).
The amount of paranitroaniline liberated was determined by reacting kallikrein with the substrate for 15 minutes at 37°C using
Measurement was performed at a wavelength of 405 nm. One unit is the amount of enzyme that decomposes 1 μmol of substrate per minute. The present invention will be explained in detail below with reference to Examples. Example 1 100 ml of Cephalose CL-4B (manufactured by Pharmacia Huain Chemicals) was activated with 1 g of cyanogen bromide at pH 11 for 10 minutes, and reacted with 5 g of 6-aminocaproic acid at 20°C for 12 hours.
A CL-4B-aminocaproic acid conjugate was synthesized. At this time, 1 ml of 6-aminocaproic acid was added to the swelling carrier.
2.0 μmol was bound per sample. Mix 3.0 g of N-hydroxysuccinimide and 2.5 g of EDC with 100 ml of this carrier, and
After reacting for a minute, dioxane, dioxane/
water (volume ratio 7:3), dioxane/water (3:7),
The carrier was washed with water and 0.2M citrate buffer (PH6.5), each in an amount 10 times the amount of the carrier. The activated carrier at this time had an activation degree capable of binding 1.8 μmol of glycylleucine to 1 ml of the swollen carrier. Add this activated carrier to 0.2M citrate buffer (PH6.5) at 100%
This was added dropwise with stirring into 100 ml of the same buffer in which 400 mg of aprotinin was dissolved.
Aprotinin was bound by reacting at ℃ for 12 hours. Bound aprotinin is 36.9n per ml of carrier.
It was mole hot. Fill a column with 100ml of this adsorbent, equilibrate it with 0.05M phosphate buffer (PH8.5) containing 0.5M salt,
Crude kallikrein solution 300U (specific activity 0.03U/A ₂₈₀ )
was passed through the column. Next, the column was sufficiently washed with the same buffer as the column until the absorbance value at 280 nm of the washing solution became 0.005 or less. After washing, the adsorbed kallikrein was eluted with 0.1M acetate buffer (PH4.0) containing 1.0M sodium chloride. The kallikrein thus obtained has a specific activity of
It showed a single band electrophoretically with a yield of 5.0 U/A ₂₈₀ and 92%. Also, the increase in purity at this time is approximately
It was 170 times hotter.

Claims (1)

[Claims] 1. 6-Aminocaproic acid is bound to a water-insoluble carrier as a spacer, and its terminal carboxyl group is reacted with N-hydroxysuccinimide to form an active ester at a rate of 1 to 3.5 μmol per 1 ml of the swollen carrier, and aprotinin is added to this. An adsorbent for purifying kallikrein that is covalently bonded.