JPS5855043A - Adsorbent of kallikrein - Google Patents

Abstract

PURPOSE:To refine kallikrein inexpensively and efficiently by using a bound lysine-insoluble carrier wherein lysine is bound to an insoluble carrier via carboxylic groups as an adsorbent for the purpose of refining of kallikrein. CONSTITUTION:An aminoalkylated insoluble carrier having free amino groups is obtained by activating insoluble polysaccharides such as cephallose, cephadex or cellulose powder with bromocyane to bind diamino compds. Lysine is acted upon the insoluble carrier in the copresence of a condensating agent such as 1-ethyl-3-carbodimide whereby the bound lysine-insoluble carrier wherein the lysine is bound to the insoluble carrier via carboxylic groups is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel adsorbent for use in the purification of kallikrein.

Kallikrein according to the present invention is also called kallidinogenase because it acts on kininogen present in serum and liberates kallidin, a circulatory system active substance, and has been widely used as a therapeutic agent for circulatory disorders since ancient times.

Kallikrein is widely distributed in the aS of mammals, such as the pancreas and submandibular line, and in body fluids such as urine and blood, but kallikrein obtained from pig pancreas is mostly used as a pharmaceutical.

In addition to kallikrein, there are various peptidases such as trypsin and chymotrypsin in the pancreas, but contamination of kallikrein with these enzymes, especially kininase, which degrades kallidin, significantly reduces the action of kallifrein as a drug. let Therefore, methods for extracting and separating kallikrein that do not contain these coexisting enzymes have been studied for a long time.

In the past, attempts have been made to fractionate and purify using ion exchangers alone or in combination.
As seen in Publication No. 4795, a method using an adsorption carrier obtained by bonding a compound with high affinity for kallikrein to an insoluble carrier has been developed, but all of these methods are too complicated to operate as industrial production methods. In addition, many problems remain, such as excessive costs for mass production and insufficient purification efficiency.

In order to solve these problems of the conventional methods, the present invention aims to develop an adsorbent for kallikrein that is inexpensive, simple and efficient in purification, and can be operated satisfactorily on an industrial scale.・Tamo? As an adsorbent used for purifying kallikrein from an aqueous solution containing kallikrein, it is characterized in that lysine is bonded to an insoluble carrier via its carboxyl group.

When using the above-mentioned adsorbent according to the present invention, the pH should be adjusted to 7-7.
A kallikrein aqueous solution adjusted to pH 9 was brought into contact with the adsorbent equilibrated with a pH 7 to 9 buffer, the adsorbent was further washed with the same buffer, and then an electrolyte was added to increase the ionic strength of the same buffer. By using a simple process suitable for industrial production, in which kallikrein is eluted with a liquid, λ
Ru.

The adsorbent according to the present invention is one in which lysine is bound to an insoluble carrier via its carboxyl group, and is prepared by a known method (for example, "ProtoinPurificati
on by Affinity Chromatogr
aphy J The Journal of Biol
logical chemistry, Vo1245
IPISO5? (197 calls issued).

That is, insoluble polysaccharides such as Sepharose (trade name, Pharmacia, Sweden), Sephadex (trade name, Pharmacia, Sweden), or cellulose powder, etc.
Activated by cyanide, diamino compounds, e.g.
By coupling ethylenediamine, an aminoalkylated insoluble carrier having free amino groups can be obtained.

In the insoluble carrier, in the presence of a condensing agent such as 1-ethyl-3-(5-')methylaminopropyl)carbodiimide,
By acting with lysine, it is possible to bind to an insoluble carrier via a carboxyl group or to obtain a sine-insoluble carrier bond.

Incidentally, a lysine-insoluble carrier conjugate can also be obtained by using aminoalkylated polyacrylamide as the insoluble carrier and performing the same procedure as described above.

Conventionally, adsorbents obtained by binding lysine and arginine to insoluble carriers have been used to purify enzymes with trypsin-like substrate specificity.
Publication No. 99191 relates to a method for obtaining kallikrein by adsorbing urinary kallikrein to arginine-Sepharose and then dissolving it, and
“Methocjs in Fjr+zymology
XLV J (issued in 1976) and Special Publication No. 53-18
No. 05 describes a method for purifying plasmin and plasminogen using lysine-Sepharose.

However, the adsorbent used in each of these methods is a conjugate obtained by reacting lysine or arginine with bromcyane-activated Sepharose.
Lysine or arginine forms a bond structure in which it is bonded to sepharose via its amino group,
Therefore, in its binding structure, it is completely different from the lysine-insoluble carrier conjugate according to the present invention. Also,
With lysine-Sepharose in the above conventional bonding configuration,
As shown in the experimental example (Table 1) below, unlike the case of urinary kallikrein, it shows only a slight adsorption property to porcine pancreatic kallikrein, and in terms of kallikrein adsorption properties, the lysine-insoluble lysine according to the present invention It is different from carrier conjugate.

In order to avoid confusion between the two above, in the following explanation, the adsorbent according to the present invention, for example, an adsorbent made using Sepharose as an insoluble carrier, will be referred to as Lysyl-Sepharose and the conventional Lysyl-Sepharose. To call. In carrying out the present invention, the aqueous kallikrein solution and the lysyl-insoluble carrier conjugate, such as lysyl-Sepharose, can be brought into contact by either a column method or a batch method.

Furthermore, binding of kallikrein to Lysyl-Sepharose is preferably carried out at a pH of 7 to 9, and buffers used for this purpose include Tris-HCl buffer, ethanolamine-HCl buffer, boric acid 11 buffer, and phosphoric acid 11 buffer. Any commonly used buffer solution may be used, but the concentration is preferably 0.02M or lower.

In addition, the carrier adsorbed kallikrein is, for example, 0.0
After washing with 2M Tris-HCl buffer, pH 8 to remove unadsorbed substances, trypsin and the like are eluted and removed with the same buffer containing an electrolyte, for example 0.3 to 0.4M NaCl, and then 0. Kallikrein is eluted with the same buffer containing 4-0.6M sodium chloride. Furthermore, instead of increasing the salt concentration stepwise, kallikrein can also be eluted by linearly and continuously increasing the salt concentration using the same buffer containing 0.1 to 0.8 M salt.

This treatment made it possible to obtain purified kallikrein with a yield of 80-90%, which is related to the purity of the starting material, but is largely free of kininase.

In addition, in the present invention, the activity measurement method shown below is used at 7°ti1.
9Ly4y-9□ G, W using benzoyl-L-arginine ethyl ester as a substrate in the presence of soybean trypsin inhibitor.
, Shwert et al.'s absorbance method (Biochem, B
iophys Acta, 16.570 (1955
)).

In addition, when IPmol of substrate was decomposed in 1 minute, this was defined as IEu.

2. Measurement of kininase activity Based on the kinin assay method (Evaluation of Medicinal Efficacy (1), p. 974, (@Ichininshokan, published in 1972)) using bradykinin (produced by the Protein Research Foundation) as a substrate, 30° After reacting for 2.5 minutes at C, the amount of remaining bradykinin was determined by contraction of the guinea pig ileum, and the kininase activity was determined by ng BK.
Expressed in decomposition/min/gu.

Experimental Example Crude kallikrein (Q, 5'FUu) obtained from pig pancreas
/'P) 50 cape to 5-102 cape Tris-HCl buffer,
Dissolve at pH 8.0, equilibrate with the same buffer, or apply to a column of Zin-Sepharose or Risyl-Sepharose (t
zxlocll) and the column was washed with the same buffer to remove unadsorbed substances.

Next, kallikrein was eluted with the same buffer containing 0.6M sodium chloride, and the adsorption rate of each was examined.

The results of the above adsorption rates are shown in Table 1 below.

Table 1> The results in Table 1 above confirm that the adsorption rate of the lysyl-Sepharose according to the present invention, that is, the lysyl-insoluble conjugate, to the aqueous solution of porcine pancreatic kallikrein is extremely low.

Example 1 Autolyzed fluid 251 obtained from 10 fresh pig pancreases
! An equal volume of 0.02 M) Lysyl-HCl buffer (pH 8.0) was added thereto, and the mixture was passed through a column packed with 21 Lysyl-Sepharose equilibrated in advance with the same buffer. After passing through the column, the column was washed with buffer solution 41, and elution was performed by linearly increasing the salt concentration using each of the same buffer solutions 21 containing 0.1M salt and 0.8M salt. Kallikrein active fractions (Q, 4-0.6M salt concentration) were collected, desalted, concentrated and lyophilized to produce 35E, u/ which contains only kininase of less than 10 ngBK decomposition/min/Eu.
19 purified kallikrein 39 was obtained.

Example 2 Autolyzed F fluid obtained from fresh pig pancreas 10I4
1! An equal volume of 0.021 g triethanolamine-hydrochloric acid buffer, pH 8.0, was added to the solution, and the mixture was passed through a column packed with 21 Lysyl-Sephadex equilibrated with the same buffer. After passing through the solution, the plate was washed with the same buffer [2/] and then with the same buffer containing 0.35 M NaCl, and kallikrein was eluted with the same buffer containing 0.5 M NaCl. The kallikrein fraction was collected, desalted, concentrated, and lyophilized to obtain 27B-, which contains only kininase of less than 10 ngBK decomposition/min/Eu.
4 g of purified kallikrein was obtained.

Example 3 10Kl of fresh pig pancreas! Autolytic liquid obtained from 25
/ Add acetone 61 and mix, then add calcium chloride 6
Add 00g and stir well to dissolve. The precipitate was collected by centrifugation, and the precipitate was 11! containing 20 ggo'rA. It was dissolved in a solution of After desalting, an equal volume of 0.02M) Lis-HCl buffer, pH 8.0, was added and equilibrated with the same buffer in advance. lysyl-cellulose column. After passing through the solution, the plate was washed with the same buffer 2/ and then with the same buffer containing 0.55M NaCl, and kallikrein was eluted with the same buffer containing 055M NaCl. Kallikrein fractions were collected, desalted, concentrated, and lyophilized to yield 10 ng BK decomposition/min/Eu.
100Eu/M purified kallikrein 0.9 containing only the following kininases! I got J.

Example 4 Autolyzed IP fluid obtained from 10 pieces of fresh pig pancreas 25
1 to 0.62M) ethanolol raremine-hydrochloric acid buffer,
Add an equal amount of pH 8.0, add Lysyl-Sepharose 31 equilibrated in advance with the same buffer, and stir for 2 hours.

The carrier was collected by filtration, thoroughly washed with 0.02M) reethanolamine-hydrochloric acid buffer, washed with the same buffer 51 containing 0.4M NaCl, and then washed with the same buffer 51 containing 0.6M NaCl. Kallikrein is eluted by extraction three times. The eluates were combined, desalted, concentrated, and lyophilized to yield 20 Bu/'9 (kininase activity -1 = 15
4.5 g of purified kallikrein (less than ng BK decomposition/min/Eu) was obtained.

Applicant: Okura Pharmaceutical Co., Ltd.

Claims (1)

[Claims] An adsorbent for kallikrein, characterized in that lysine is bonded to an insoluble carrier via its carboxyl group.