JPH01240185A - Method for purifying angiotensin converting enzyme - Google Patents

Abstract

PURPOSE:To obtain an angiotensin converting enzyme (ACE) with a high purification degree, by linking the ACE in a sample to immobilized iminodiacetic acid groups and then liberating the ACE. CONSTITUTION:In order to bind iminodiacetic acid groups linked and immobilized on the surface of a beadlike carrier, such as glass, to ACE, the iminodiacetic acid groups are previously bound to zinc ions, such as zinc chloride. Excess zinc ions unbound to the iminodiacetic acid groups are then separated. A lung of a rabbit, etc., is subsequently homogenized, etc., and centrifuged to provide a cell membrane fraction, etc., which is then adjusted to afford an ACE-containing sample. The resultant sample is then brought into contact with the iminodiacetic acid groups linked to the zinc ions. The ACE bound to the iminodiacetic acid groups is subsequently liberated and eluted with an eluting solution containing a chelate antagonistic agent, such as glycine. Thereby, the aimed ACE with a high purification degree can be obtained by simple operation. The above-mentioned method is more preferably carried out by especially using column chromatography.

Description

Detailed Description of the Invention (Industrial Application Field) Angiotensin-converting enzyme (hereinafter abbreviated as ACE) acts on angiotensin I in vivo to dehydrate the histidyl-leucine dipeptide at its carboxyl terminal, converting it into angiotensin I. It is an enzyme that converts This angiotensin ■ acts on receptors in peripheral arteries to constrict blood vessels and promote an increase in blood pressure, and also acts on the adrenal cortex to promote the secretion of aldosterone, which is a very important physiological activity. ACE also acts on bradykinin. It also has the function of deicing phenylalanine-arginine and serine-proline dipegtide and inactivating pradikinin.

Thus, ACE is an enzyme that plays an important role in the renin-angiotensin system and the kinin-kallikrein system.

The present invention relates to a method for purifying this ACE.

(Prior Art) As a method for purifying ACH, for example, analytical biochemistry (Analytical Biochemistry) is used.
istry, Vol. 111, pp. 227-234, 1981) (hereinafter referred to as the first method), the method described in the Journal of Biological Chemistry (Jour.
nal of Biological Chemistry
try volume 257, pages 14128-14133, 1982
(hereinafter referred to as the second method), the method described in Placenta (Vol. 6, pp. 543-549, 1985) (hereinafter referred to as the third method), etc. There is.

The first method involves obtaining ACE-containing polyethylene glycol precipitate from human serum, treating this precipitate suspension with an ion exchanger to roughly purify ACE, performing polyethylene glycol precipitation again, and then This method uses hydroxyapatite to adsorb the ACE inside.

The second method involves treating rabbit testes with a surfactant to
A sample containing E was obtained, and after streptomycin sulfate was added to this sample to remove nucleic acids, N-α[1-(S)-hydroxy-3-phenylpropyl]-L-lysyl-monoproline was bound. This method involves adsorption to a resin and further gel filtration.

The third method is to solubilize a membrane fraction prepared from human placenta by trypsin treatment, adsorb it onto a captogril-bound resin, and then perform gel filtration to purify ACE.

(Problems to be Solved by the Invention) Conventionally used ACB purification methods have several problems. For example, in the first method, two steps of polyethylene glycol precipitation, ion exchange chromatography, and hydroxyl There are problems such as apatite adsorption, which requires complicated and time-consuming operations. For example, in the second and third methods, a substance that can act as an ACE inhibitor, such as N-α[1-(S)-hydroxy-3-phenylglopyl]-L-lysyl-proline or captogril, is used. In affinity chromatography using A, if contaminant proteins such as albumin coexist with ACE,
For example, the purification of CE tends to be incomplete.

As a result of intensive studies on the above-mentioned problems, the present inventors have solved these problems by utilizing the affinity between iminodiacetic acid groups and ACE, and have completed the present invention.

That is, the present invention provides the following steps: (a) binding the immobilized iminodiacetic acid group and zinc ion, (b) removing unbound zinc ions, and (c) subsequently attaching angiotensin converting enzyme in the sample to the iminodiacetic acid group. (d) removing impurities in the sample; (e) then releasing the bound angiotensin-converting enzyme;
The present invention provides a method in which the operation c) is carried out in a column packed with iminodiacetic acid groups immobilized on an organic or inorganic carrier. Further, the present invention combines the operations (a) to (e) above with at least one type of chromatography selected from affinity chromatography using an ACE inhibitor, gel filtration chromatography, and ion exchange chromatography. It also provides a method to do so.

The present invention will be explained in detail below.

(Means for solving problems) Examples of samples containing ACF include rabbits and pigs.

It may be obtained by homogenizing human lungs, placenta, testes, etc., and then centrifuging them to prepare both cell membranes. Examples of preparation methods include treatment with enzymes such as trypsin, treatment with Triton, etc. There are no limitations as long as the method does not deactivate ACE, such as a surfactant treatment method such as (Triton) X 100 or a freeze-thaw method.

The iminodiacetic acid group is used to separate ACF and its impurities.
Immobilize it by chemically bonding it to solids and m.

At this time, the chemical bond between the iminodiacetic acid group and the solid phase may be any chemical bond that does not interfere with the chelate bonding of the iminodiacetic acid group with the zinc ion.

The solid phase base material is not limited as long as it is used in ordinary biochemical reactions, such as glass, inorganic base materials such as silica, styrene base materials, and ethylene glycol base materials.

Polymers such as methacrylates, copolymers thereof, and polysaccharides such as dextran, agarose, and starch may be used.

The present invention may be carried out by constructing a reaction vessel using, for example, the above-mentioned base material, and immobilizing the imino di# acid group on the entire inner wall as a solid phase. It is preferable to construct a bead-shaped carrier using a base material, bond iminodiacetic acid groups to the surface of the carrier, and fill a column with these carriers.

Column chromatography in which a column is packed with a carrier to which iminodiacetic acid groups are bonded will be described below.

The iminodi# acid group and ACE bond in a 1:1 ratio. Therefore, if the ACE concentration in the sample is not expected, it is sufficient to use a large amount of iminodiacetic acid groups.In the present invention, in order to bond the iminodiacetic acid groups and ACE, zinc ions are added to the iminodiacetic acid groups in advance. The interaction between the iminodiacetic acid group, zinc ion, and ACE is not clear.8According to the study by the present inventors, in the case of cobalt ions, the bonding of ACH to the iminodiacetic acid group occurs at t11.
It wasn't noticed.

To bond zinc ions with iminodiacetic acid groups, for example, a zinc chloride (ZnCβ2) solution or the like may be added to the column. Zinc ions and iminodiacetic acid groups form a chelate bond in a 1:1 ratio. Therefore, the zinc ions used need only be in excess relative to the iminodiacetic acid groups.

Next, the zinc ions of the barb that are not bound to the iminodiacetic acid groups are separated. This can be done by adding to the column a cleaning solution that does not release the zinc ions bonded to the imino-diacid groups.

Subsequently, the ACE-containing material prepared as described above is brought into contact with the iminodiacetic acid group bound to the zinc ion. This can be done by adding a sample to the column.At this time, although the interaction between iminodiacetic acid groups, zinc ions, and ACH is not clear, it is important to allow the reaction between them to equilibrate. The time required for equilibration, which is good for yield etc., varies depending on the ACE concentration in the sample, the column liquid temperature, etc. This may be investigated by measuring the activity of ACH in the fractions eluted without the use of ACH.

In the manner described above, ACB bound to the iminodiacetic acid group is released and eluted from the column. To release ACE bound to iminodiacetic acid groups, an elution solution containing a chelate antagonist such as glycine may be used. If contaminants are expected to be present,
It is best to take advantage of the difference in the affinity of the impurities and ACE for the iminodiacetic acid group. For this purpose, when adding the elution solution as described above to the column, apply a concentration gradient of the chelate antagonist to the elution from the column. The chelate antagonist concentration gradient, which can be obtained by fractionating the liquid, can be linear (so-called gradient ends) or stepwise (so-called steps), and can be obtained by using ACE with a higher degree of purification. In order to obtain this, it is preferable to reduce the fractional volume of the eluate and elute ACE with an elution solution that provides a gentle chelate antagonist concentration gradient, within the range of the chelate antagonist concentration gradient.

Conditions such as the fraction amount may be determined by conducting preliminary experiments in consideration of the sample to be used.

The activity of ACE is described, for example, in the Journal of Chromatography 4-(Journal of Chromatography 4-).
233 volumes.

It may be measured according to the method of Horiuchi et al. described on pages 123 to 130.

The method described above may be used in combination with conventionally known methods such as affinity chromatography using an ACE inhibitor, gel r perchromatography, and ion exchange chromatography. It may be carried out prior to the present invention using the present invention, or it may be carried out using the AGE fraction obtained in the present invention as a sample.

(Effects of the Invention) According to the present invention, ACE with a high degree of purification can be obtained by simple operation.
can be obtained. In particular, if the present invention is carried out as column chromatography, ACE can be purified more easily and quickly.The present invention utilizes the affinity between ACE and iminodiacetic acid groups, but other physical properties of ACB can be used. By combining chromatography utilizing its properties with the present invention, it is possible to obtain ACE with a higher degree of purification.

EXAMPLES Hereinafter, examples will be shown to explain the present invention in more detail, but the present invention is not limited to these examples.

(Example) After cutting human placenta 1kIr into pieces, 0.9% of 41
Wash the blood by immersing it in NaCβ solution and add 0.25M of 2J2.
20mM potassium phosphate #1 street solution containing sucrose (pH 7)
, 8).

This was centrifuged at 700xg for 30 minutes at 4°C to collect the supernatant. The pH of the supernatant was adjusted to 5.2 using 0.1M acetic acid and incubated for 5 minutes. After that, 15.000xg,
The membrane fraction was collected as a precipitate by centrifugation at 4°C for 30 minutes.

The membrane fraction was prepared using 20mM potassium phosphate buffer (pH 7, 8).
).

The protein amount of the membrane fraction at this time was 3810 ng.
゛The amount of protein is Lowry (Lowry)
Journal of Biological
Chemistry vol. 193, p. 265 (1951
(see 2013).

To the membrane fraction, 7.62μ trypsin was added in the presence of 1mM CaCβ2, and after incubation in a 37°C water bath for 120 minutes, centrifugation was performed at 15,000xg for 30 minutes at 4°C, and the soluble fraction was added to the supernatant. Collected the amount. The protein content of the soluble fraction is 2,640 μ, and the ACE activity is 49.8
5μmo! It was 2/min. Using this soluble fraction as a sample, ACE was purified by ion exchange chromatography using a DEAE group, affinity chromatography using captogril, and the method of the present invention. First, this soluble fraction was pre-equilibrated with 20mM potassium phosphate buffer (pH 7.8), and DEA was added.
E-Toyopearl 650s (manufactured by Tosoh) was added to a column (1.5 x 15an) packed with the same buffer at 1.01%.
Washed with. After that, the adsorbed ACE is 0-0.5M
A linear concentration gradient (500 ml) of NaCβ was used to elute and fractionate 7 ml each. E activity is N a C
It was eluted with a peak at a concentration of 125 mM/l. Both parts were added to 20mM potassium phosphate buffer (pH 7, 8).
It was dialyzed and desalted.

Next, captopril 60■ was AH
- Captopril-AH-Sepharose 4B (1,OX6.0Gl) bound to Sepharose 4B (Pharmacia)
Affinity column chromatography was performed using 1). 20mM potassium phosphate buffer (
The above fraction was added to a column equilibrated with p) ('7.8) and washed with 200 ml of the same buffer. Thereafter, the adsorbed ACE was eluted and fractionated by J cQl using a linear concentration gradient (200 ml) of 0 to 0.5 M NaCl, and the ACE activity was eluted with a peak near the NaCl concentration of 250 mM. After concentrating this fraction, TSK-gel Chelat with iminodiacetic acid group was added.
e-5PW (manufactured by Tosoh) < 7, 5 mm
7,5 cm) for further purification. Add 100 μmoβ Z n C12 solution in advance,
In addition, 20mM Tris-HCl containing 0.5M NaCl? The above sample was added to a column that had been equilibrated with R solution (pH 8,0), and the same gentle W! After washing with solution, the adsorbed ACE was washed with a linear concentration gradient of 0 to 0.2 M glycine (60
ml) to elute and fractionate in 4 ml portions.

Note that the flow rate was 1 ml/min. ACE activity is
It was eluted with a peak around the glycine concentration of 50 mM.

The amount of protein in the obtained AGE fraction was 0.53■,
The specific activity was 39.51 μmoβ/min/q. This AGE fraction showed a single band in 5DS-polyacrylamide electrophoresis. The total activity (μ
mo1/m1n), total protein amount (■), specific activity (μmo
Table 1 shows the purification degree of ACH, that is, the ACE purity (times), when the value after solubilization treatment is set as 1, which was determined based on β/min/■) and specific activity.

Claims (3)

[Claims] (1) (a) Bind the immobilized iminodiacetic acid groups and zinc ions, (b) Remove unbound zinc ions, and (c) Subsequently bind angiotensin converting enzyme in the sample to the iminodiacetic acid groups. (d) removing impurities in the sample; and (e) then releasing the bound angiotensin converting enzyme. (2) A method according to claim (1), characterized in that a column packed with iminodiacetic acid groups immobilized on an organic or inorganic carrier is used. (3) In the method of claim (1) or (2), one or more types of chromatography selected from affinity chromatography, gel filtration chromatography, and ion exchange chromatography using an angiotensin converting enzyme inhibitor is performed. A method for purifying angiotensin converting enzyme, characterized by: