JPS6137095A - Method of separating enzyme analog to trypsin - Google Patents

Abstract

PURPOSE:To separate an enzyme analog to trypsin and/or its precursor by liquid chromatography, by using a totally porous adsorbent containing an alcoholic hydroxyl group as a stationary phase. CONSTITUTION:A totally porous adsorbent having an alcoholic hydroxyl group and a ligand having affinity for an enzyme analog to trypsin is used as a stationary phase of chromatography. 1.0-14.0 millimol/g, preferably 1.5-11.0 millimol/g based on dried weight of the adsorbent of alcoholic hydroxyl group exists in the adsorbent. The ligand group on the adsorbent is fixed with a covalent bond, and 0.001-3,000mumol/g, preferably 0.001-250mumol/g ligand group exists. The ligand group on the adsorbent contains an amidino group or a guanidino group.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for separating trypsin-related enzymes and/or their precursors.More specifically, the present invention relates to a method for separating trypsin-related enzymes and/or their precursors by chromatography. The present invention relates to the separation method described above, characterized in that a completely porous adsorbent is used in the method of separating precursors.

Trypsin-related enzymes are hydrolytic enzymes that have specificity for ester bonds in addition to peptide bonds of arginine and lysine residues.The above enzymes include trypsin, plasmin, urokinase, thrombin, and the like.

In fields such as biochemistry and medicine, separating and analyzing the above enzymes and/or their precursors from mixtures containing them is
This is one of the important issues.

For example, trypsin is an enzyme that is specifically produced in the pancreas, but it escapes into the blood when pancreatic disorders occur, so measurement of trypsin in serum is considered to have great significance in diagnosing pancreatic diseases.

In addition, we also know the amount and status of enzymes involved in blood coagulation and fibrinolysis, such as plasmin, urokinase, and thrombin, as well as plasminogen, the precursor of plasmin, in body fluids in some diseases. This has become extremely important.

[Conventional technology]

Many methods are currently used to separate the enzyme and/or its precursor.

For example, l) Method using solubility difference 1. (3) a method that utilizes a difference in molecular size or shape; and (3) a method that utilizes a difference in chemical or physical affinity. Furthermore, separation methods that utilize biologically specific affinity have high selectivity and are widely used. In particular, affinity chromatography, in which one of the substances exhibiting affinity (hereinafter referred to as a ligand molecule) is immobilized on an insoluble carrier and the other is selectively separated, is widely used due to its operability (Chibata, Tosa (See “Affinity Chromatography” by Tetsuya and Yushi Matsuo, Kodansha).

Hitherto, agarose, in particular, has been widely used as an insoluble carrier for Affinity chromatography (for example, trade name Sepharose, Pharmacia, Sweden). However, agarose has the following drawbacks.

[Problem that the invention seeks to solve]

That is, agarose has many operational limitations because it can hold an extremely large amount of water and has insufficient strength when wet. For example, they are susceptible to destruction during operations such as activation, immobilization, etc.

In addition, for example, it is important to know the amount present in body fluids, but when analyzing the above enzymes to aid in the diagnosis and treatment of pathological conditions, it is important to be able to analyze with a small amount of sample, to be quick, and to be simple. required. However, in conventional affinity chromatography, for example, when agarose is used, its strength is insufficient, so when it is packed in a column, it is not possible to flow the liquid containing the substance to be separated at a high flow rate. Continuous detection of separated components at the same time as elution has disadvantages, such as the difficulty of detecting separated components.
It lacks convenience.

Therefore, as a result of intensive studies to resolve the above-mentioned drawbacks of conventional affinity chromatography, we developed a method for quickly and conveniently separating and analyzing the enzymes mentioned above mainly by using high-speed affinity chromatography, which led to the present invention. Ta.

[Means for solving problems]

That is, the present invention uses a fully porous adsorbent having an alcoholic hydroxyl group and a ligand group having affinity for the enzyme as a stationary phase in a method for separating trypsin-related enzymes by liquid chromatography. The present invention relates to a method for separating trypsin-related enzymes, which is characterized by the following.

Trypsin-related enzymes in the present invention include not only normal trypsin-type enzymes but also their precursors. Ordinary trypsin-type enzymes are hydrolases that have specificity for ester bonds as well as peptide bonds of arginine, 1- and lysine residues, such as trypsin, plasmin, urokinase, and thrombin. etc. In addition, its precursor is a substance that has not yet become a normal trypsin-type enzyme in vivo, for example,
Examples include trypsinogen and plasminogen.

A fully porous adsorbent having an alcoholic hydroxyl group and a ligand group having an affinity for trypsin-related enzymes is used as the stationary phase in the adhesive pmography of the present invention. The alcoholic hydroxyl group in the adsorbent is present in the range of /, 0-/4/, θmmol/f per dry weight of the adsorbent. Since the alcoholic hydroxyl group is contained within this range, the filler made of this adsorbent has hydrophilic properties and does not show hydrophobic adsorption or distribution to many water-soluble substances in water. The amount of hydroxyl groups is practically t! ~t/, Ommol/f
It is good that it is within the range of . Among the alcoholic hydroxyl groups, hydroxyl groups derived from vinyl alcohol units are particularly preferred.

The amount of hydroxyl groups can be determined by reacting the hydroxyl groups with acetic anhydride and measuring the amount of acetic anhydride consumed or the change in weight of the adsorbent. At this time, if the functional groups on the carrier for adsorbing biological components also react, the reaction can be determined by the above method after protecting the functional groups. Let /mmol/r be the amount of hydroxyl groups when the dried adsorbent /f reacts with /mmol 1 of acetic anhydride.

In the physical property measurements described below, if the functional groups on the adsorbent react under the measurement conditions, the physical properties can be determined by protecting the functional groups and measuring them in the same way as when measuring the amount of hydroxyl groups.

The ligand groups on the adsorbent are immobilized by covalent bonds, with o,o O/ ~3o o o μmol/f%
Preferably, it is present in a range of 0.001 to 230 μmol/f. By the presence of the ligand group in this range,
The adsorbent used in the present invention is a suitable packing material for high-speed affinity chromatography.

The ligand group on the adsorbent used in the present invention has an amidino group or a guanidino group. In addition to having affinity for trypsin-related enzymes, the ligand group also has properties such as: 1) being chemically stable; 2) having no or little non-specific interactions; desired. It is desirable that the ligand molecule has, in addition to the ligand group, a suitable functional group that can be utilized for the immobilization reaction. Preferably, a compound having a structure represented by the following formula is desired.

A is a phenyl group or a suitable aliphatic compound, and R is a nucleophilic reactive group having active hydrogen such as an amino group, a carboxyl group, or a hydroxyl group. ] Specific examples of ligand molecules include m- or p-aminopenzamidine, m-4* is p-aminophenylguanosine, arginine, Hiro-aminobutylguanidine, t
trans-#-aminobutylguanidine, trans-
41-amidinomethylcyclohexanecarboxylic acid, tr
It is selected from ans-II-guanidinomethylcyclohexanecarboxylic acid, trans-≠-aminomethylcyclohexanecarboxylic acid, or lysine, and may be a combination of two or more.

The ligand group is covalently bonded to the active group either directly or via a spacer. The active group refers to the amino group, carboxyl group, hydroxyl group of the ligand molecule or spacer molecule,
Refers to a group that can undergo substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as a thiol group, and specifically includes an imidazolyl carbamate group, an imidocarbonate group, a cyanate ester group, an epoxy group, a carbonate group, and a bromoacetyl group. , a halogenated triazine group, etc., preferably an imidazolyl carbamate group, an imidocarbonate group, a cyanate ester group, an epoxy group, etc., and an imidazolyl carbamate group is particularly preferred. A spacer has a structure in which hydrophilic functional groups that are not strongly ionic groups at both ends of a linear molecule are covalently bonded to a ligand molecule and an active group, respectively, so that the ligand group and the target substance are sufficiently connected. Preferably, the length is such that they can interact.

The linear molecule has, for example, a methylene chain, an amide bond, an ether bond, etc. between the hydrophilic functional groups at both ends, and has a molecular length corresponding to the number of carbon atoms / to /θ. Specifically, t-aminohexanoic acid, β-alanine-/, /, /, J-diaminohexane, bis(3-
Aminopropyl) amine, glycine, glycylclycine, chrysylglycylglycine, succinamidoethylamine, succinamidobutylamine, aminoethanol, 2. ! -diaminodiethyl ether etc. Preferred are t-aminohexanoic acid, J-diaminohexane, glycylglycine, and the like. A single type of spacer or two or more types of spacers may be used together.

The amount of immobilized ligand groups and spacers can be determined as follows. For example, if the ligand molecule is a substance that absorbs in the ultraviolet and/or visible regions, the carrier for adsorbing biological components can be hydrolyzed with tN hydrochloric acid, etc., and then the absorbance of the supernatant can be measured. The amount of immobilized ligand groups can be determined. At this time, for example, if the above linear molecule is a substance having an amino group,
The amount of immobilized spacer can be determined by analyzing the above supernatant using an amino acid analyzer or the like. A simpler method for determining the amount of immobilized ligand groups is to collect the ligand molecules that remain unimmobilized during the immobilization reaction of the ligand molecules, and determine the amount by measuring absorbance, etc. It is also possible to estimate the amount of immobilized ligand groups by determining . In addition, as a more convenient method for determining the amount of immobilized spacer, at the end of the spacer,
If a titratable functional group such as an amine group or a carboxyl group is present, there is also a method of determining it by titration at the stage where the spacer is immobilized.

The adsorbent used in the present invention is made of a crosslinked copolymer. The crosslinked structure is not particularly limited, but a structure crosslinked by a crosslinkable monomer unit having a triazine ring, or a structure formed by bonding epichlorohydrin or a bisepoxy compound to a hydroxyl group is preferable. Among these, structures crosslinked by crosslinkable monomer units having a triazine ring, such as triallyl isocyanurate and triallyl cyanurate, are preferred. A crosslinkable monomer unit having a triazine ring is
It represents a structure formed by polymerization or copolymerization of monomers represented by the following formulas (4) and (B).

(A) (B) (However, R1, R2 and R3 are each independently -C
H2CH=CH2, -CH2c=ca or -CH2-C
= indicates CH2. )CH3 Among these, triallylisocyanurate in which RI XR2 and R3 are all -CH2CH=CH2 in formula (4) is particularly preferred.

The proportion of crosslinkable monomer units in all monomer units forming the adsorbent is preferably within the range expressed by the following formula (1).

(However, a is the mole fraction of monomer units excluding crosslinkable monomer units in the adsorbent, b is the mole fraction of crosslinkable monomer units,
n is the number of functional groups such as polymerizable vinyl groups that the seven molecules of the crosslinkable monomer have. ) When high mechanical strength is required, such as a packing material for high-speed affinity chromatography, it is more preferable that the left side of equation (1) is 0.2.

In order to balance the separation ability and strength of the adsorbent filler, it is necessary to maintain the water retention amount of K within an appropriate range. Conventionally, fillers such as cross-linked agarose and cross-linked dextran have high water content and low mechanical strength, as described above, and this tendency is particularly large for fillers with large pore sizes. The filler obtained from the adsorbent used in the present invention has a water content of O1! regardless of the pore size. It is in the range of ~1 Aot7, preferably O1j ~3.0 t/l, and has sufficient strength to be used as a packing material for high-speed affinity chromatography. The water retention capacity of an adsorbent is expressed as the amount per adsorbed weight.The copolymer that has been sufficiently swollen in water is placed in a centrifuge tube equipped with a filter, and the water adhering to the surface of the copolymer is centrifuged. It can be determined by drying the aggregate and determining the weight change before and after drying.

The specific surface area of the adsorbent is usually j ~ / 000 rr? /
It is in the range of f. In other words, the adsorbent used in the present invention has permanent bores and is hard, so when it dries, the bores shrink somewhat but maintain almost their swollen state.

Although there are various methods for measuring the specific surface area, in the present invention, the most common method is the BET method using nitrogen gas.

In addition, the sample used for specific surface area measurement must be sufficiently dried. The carrier of the present invention has high hydrophilicity and is difficult to dry, so after equilibrating the water-wet carrier with acetone,
It is preferable to dry under reduced pressure at a temperature below 0°C.

The shape of the adsorbent used in the present invention is not particularly limited, and can take any shape such as granules, membranes, threads, and lumps depending on the method of use. When used as a filler for high-speed affinity chromatography, it is preferably granular or spherical. In that case, the particle size is not particularly limited, but the weight average particle size is usually 3~! It is in the range of OOμm. When used as a packing material for high-speed affinity chromatography, -7μ-3~20 pm, more practically 3~/! pm
It is preferable that it is in the range of .

Next, an example of a method for manufacturing the adsorbent used in the present invention will be described.

In the adsorbent used in the present invention, for example, 70 to ♂O of the ester groups of a copolymer consisting of a carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring are converted into hydroxyl groups by saponification or transesterification. After converting,
By directly reacting and immobilizing a ligand molecule with the active group of an activated carrier obtained by reacting a part of the hydroxyl group with an activation reagent, or by directly reacting and immobilizing the active group of the activated carrier with a spacer molecule. It can be obtained by reacting with the carrier to form a spacer molecule-attached carrier, and then immobilizing a ligand molecule at the end of the spacer molecule on the spacer molecule-attached carrier. Alternatively, a spacer molecule and a ligand molecule are reacted in advance to synthesize a compound in which the ligand molecule is covalently bonded to the end of the spacer molecule, and the compound is reacted with the active group of the activated carrier. It is also possible to obtain it by

Here, the carboxylic acid vinyl ester refers to a compound having one or more polymerizable carboxylic acid vinyl ester groups, and is selected from vinyl acetate, vinyl propionate, vinyl butyrate, vinyl valerate, and vinyl pivalate. Alone again V! ,, used in combination of two or more types. Among them, vinyl acetate and vinyl propionate are particularly preferred from the viewpoint of ease of polymerization, transesterification, saponification, and availability.

In addition, the crosslinkable monomer having a triazine ring is one represented by the above-mentioned formula (Al straddling (Bl). Among them, in (4), R1, R2 and R3 are all CH2
Triallylisocyanurate, which is CH""CI (2), is preferred as a crosslinking agent because it has good copolymerizability with vinyl acetate and high stability against transesterification or saponification.

The polymerization to obtain a copolymer composed of a vinyl carboxylic acid ester and a crosslinkable monomer having a triazine ring can be carried out by a conventional polymerization method such as suspension polymerization, bulk polymerization, or emulsion polymerization. Suspension polymerization is preferred when obtaining fillers for high speed affinity chromatography.

In order to obtain the adsorbent used in the present invention, it is important to copolymerize monomers other than carboxylic acid vinyl ester and crosslinkable monomers having a triazine ring to the extent that they hardly affect the physical properties of the copolymer. There is no problem.

In addition, when copolymerizing a crosslinkable monomer having a carboxylic acid vinyl nystertotriazine ring, a copolymer obtained by adding one or more organic solvents that dissolve the monomer to the monomer. Along with forming a permanent bore,
Control the pitting corrosion, pore size or pore size distribution of the bore. Organic solvents that dissolve monomers include aromatic hydrocarbons such as toluene and xylene, aliphatic hydrocarbons such as heptane, octane, cyclohexane, and decalin, and acetic acid.
-butyl, lIo-butyl acetate, n-hexyl acetate,
These include aliphatic esters such as dioctyl adipate, aromatic esters such as dimethyl phthalate, dioctyl phthalate, and methyl benzoate, and alcohols such as butanol, heptanol, and octatool. When carrying out suspension polymerization, a solvent that is difficult to dissolve in water is preferred. These organic solvents are used in an amount of 20 to 300 parts by weight based on 100 parts by weight of the monomer. In particular, when producing a filler for high-speed affinity chromatography that requires mechanical strength, the amount of organic solvent is preferably in the range of 30 to 100 parts by weight.

Linear polymers or rubbers that are soluble in the monomer mixture may be added to the monomer mixture to control the pore size and pore size distribution of the adsorbent or to increase the flexibility of the adsorbent. Linear polymers and rubbers that dissolve in the monomer mixture include, for example, polyvinyl acetate, polystyrene, chloroprene rubber, butadiene rubber, etc.
It is used in an amount of not more than 10 parts by weight, preferably not more than 10 parts by weight.

The type and amount of the initiator used during polymerization can be selected depending on the polymerization method. In normal suspension polymerization and bulk polymerization, common radical polymerization initiators, such as 2.2'-
Azo-based initiators such as azobisisobutyronitrile, -/♂-λ, 2'-azobis-C,24-dimethylvaleronitrile), and peroxide-based initiators such as benzoyl peroxide and lauroyl peroxide. can be used.

Transesterification or saponification of the copolymer obtained by polymerization is carried out using an acid or alkali in water, alcohol, or a mixture thereof as a solvent. However, in order to obtain a filler with sufficient mechanical strength, the ratio of ester groups to hydroxyl groups, that is, the reaction rate, must be 10-♂o.
It is best to control it within a range of %. The reaction rate can be controlled by selecting appropriate conditions by changing the reaction solvent, reaction temperature, or reaction time.

Examples of methods for obtaining activated carriers by introducing active groups into copolymers obtained by saponification and/or transesterification include the following methods. The hydroxyl groups of the copolymer obtained by saponification and/or transesterification are reacted with, for example, /,/'-carbonyldiimidazole to obtain an activated carrier having imidazolyl carbamate groups.

The amount of imidazolyl carbamate groups bonded can be controlled by the amount of /, /'-carbonyldiimidazole, reaction temperature, etc.

In the case of immobilizing spacer molecules, the spacer molecule-attached carrier is obtained by reacting the active group of the activated carrier obtained as described above with the spacer molecule in the following manner. That is, by adjusting a solution of spacer molecules to an appropriate temperature, adding the above-mentioned activated carrier to the solution, suspending, and stirring, x) to obtain a carrier with spacer molecules immobilized thereon. I can do it.

As a method for immobilizing a ligand molecule on the thus obtained spacer molecule-attached carrier, the following method can be mentioned.

As an example, a carrier on which p-aminopenzamidine is immobilized as a ligand molecule can be obtained by the following method.

A carrier with a spacer molecule having a carboxyl group at the end of the immobilized spacer molecule is obtained, and a suspension containing the carrier, a ligand molecule, and a condensing agent such as carbodiimide is adjusted to an appropriate... and stirred. It can be obtained from KotoK.

Next, an example of the separation method of the present invention using the adsorbent thus obtained will be described.

A sample containing the enzyme is injected while passing a solvent serving as a mobile phase through a column filled with the adsorbent, and after the enzyme is selectively adsorbed to the adsorbent, the mobile phase is changed to another solvent. The enzyme can be separated and eluted from the column by passing a liquid through it to elute the enzyme from the adsorbent. Confirm the eluted enzyme using a detector and recorder. A series of these operations can be performed using a conventional high performance liquid chromatograph.

The shape, size, and material of the column are not particularly limited;
A stainless steel or glass column commonly used in liquid chromatography may be used. The solvent for the mobile phase is usually an aqueous solution in which salts and buffers are dissolved, and its composition can be selected depending on the sample and stationary phase. Specific examples of salts and buffers include sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, sodium acetate,
Acetic acid, hydrogen phosphate/sodium, hydrogen phosphate, 2
/-Thorium, tris hydrochloride, etc., which are usually used when separating biological components by aqueous solvent liquid chromatography, can be mentioned. The mobile phase used to elute the enzyme from the adsorbent is different from the mobile phase used before elution, for example, /
2) added a protein denaturant such as urea or guanidine hydrochloride; and 3) added a competitive inhibitor of adsorbed enzymes.

It is also possible to use a combination of two or more types of eluent solvents, changing them continuously or stepwise in multiple stages. It is also possible to initially use a mobile phase that does not allow the injected enzyme to be adsorbed to the stationary phase, but rather causes it to be eluted with a delay due to weak interaction with the adsorbent.

The eluted components can be continuously detected at the same time as they are eluted using an ultraviolet absorption or fluorescence detector, and recorded using a recorder. The presence or absence of enzymatic activity and the magnitude of the activity of the eluted component can be determined almost simultaneously with the elution of the component by the following method. That is, by mixing and reacting the eluate after exiting the above-mentioned detector with a substrate specific for the target enzyme cocoin in the flow path, for example, a synthetic substrate bound to a fluorescent substance can be produced. When the fluorescent substance is used and mixed, the released fluorescent substance can be detected by a fluorescence detector.

〔Effect of the invention〕

The adsorbent used in the present invention is hard, and when used as a packing material for high-speed affinity chromatography, it allows the eluent to flow at a high flow rate, making rapid analysis possible. In addition, the adsorbent used in the present invention is stable over a wide range, and has the advantage that it can be stably used without deterioration even under alkaline conditions, which is not applicable to adsorbents with a silica gel skeleton, for example. . Furthermore, the adsorbent used in the present invention has sufficient hydrophilicity because it has a large amount of hydroxyl groups, and has the advantage that hydrophobic adsorption to biological components and the like is slightly low.

Since the separation method of the present invention uses high-speed affinity chromatography using the above-mentioned adsorbent as a packing material, trypsin-related enzymes can be easily and quickly separated and analyzed. According to the separation method of the present invention, it is possible to specifically separate Glu-plasminogen and LIyB-plasminogen, which are the main forms of plasminogen, which is a precursor of plasmin, in blood. Applications such as diagnosis of blood coagulation and fibrinolytic diseases and purity testing of various plasminogen samples are possible.

As described above, the separation method of the present invention has made a significant contribution to the fields of biochemistry and medicine.

〔Example〕

The present invention will be explained in more detail in the following examples, but the present invention is not limited to the examples at all.

Example/ Vinyl acetate 100? ,) Realyl isocyanurate group 2, n-butyl acetate solution, decaling and, 2. ．． 2'-Azobisisobutyronitrile 3. ≠ 1 and water gOO− in which a small amount of polyvinyl alcohol and sodium phosphate were dissolved were placed in a three-necked flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirring bar, and stirred thoroughly. , otsu! ℃/r waiting time additional 7
Suspension polymerization was carried out by heating at 5° C. for 5 hours to obtain a granular copolymer. Next, pass 1p, wash with water, then extract with acetone, and add caustic soda 6! The copolymer was saponified by stirring for 2θ hours at 71°C in a 9-jl three-necked flask equipped with a reflux condenser, a nitrogen inlet tube, and a stirring bar together with methanol in which T was dissolved, and the particles were filtered. Washed with water and further dried.

The particles 10f were placed in a 5OO- beaker and dried with Molecular Sheep 4ZA!Dioxane! 00ml and 3.211f of he/'-carbonyldiimidazole were added and the reaction was carried out at room temperature for 1j minutes with stirring to obtain an activated carrier having an imidazolyl carbamate group as an active group. The activated carrier was washed with the above dry dioxane and then suction-filtered. The activated carrier was dissolved in a 1M aqueous sodium carbonate solution containing t-aminohexanoic acid λA, 2f (
By adding 2θOdK (pH 10, 0) and reacting with 2 for an hour at 3°C while shaking, 2-
A carrier with spacer molecules on which aminohexanoic acid was immobilized was obtained. The amount of spacer molecules immobilized on the su-, lk-spacer molecule-attached carrier was ≧θμrlol/f spacer molecule-attached carrier.

Place 2f of the dried carrier with spacer molecules obtained above in a 30tttl beaker, and
, o, xyr2- (N-morpholino) ethanesulfonic acid aqueous solution (...lA7o)/jtd, /-ethyl-3-(
3-dimethylaminepropyl)carbodiimide hydrochloride 0
．． 27f was added and stirred at room temperature for 30 minutes.

p-aminopenzamidine hydrochloride, 2J:
After adding lrf and adjusting the volume to 4L and 7jtK, the suspension was transferred to a 700-Erlenmeyer flask and shaken at room temperature for 2J hours to dissolve t-aminohexanoic acid as a spacer molecule and p- as a ligand molecule. A carrier for adsorbing biological components containing aminobenzamidine was obtained. The obtained biological component adsorption carrier was washed in the following order: water, a 7M aqueous sodium chloride solution containing 0.0jN sodium hydroxide, a 7M aqueous sodium chloride solution containing o,osH hydrochloric acid, and water. The solid amount of p-aminobenzamidine calculated from the absorbance at nm after collecting the washing solution was 3μ per dry biological component adsorption carrier.
It was mol. In addition, the average particle size of the carrier for adsorbing biological components is 5! Oμm, hydroxyl group density is Hiroshi mmo! /? Carrier for adsorption of biological components, water holding capacity is /, 9 f water/2 Carrier for adsorption of biological components, specific surface area is 4'Orr? /f It was a carrier for adsorbing biological components.

The adsorbent obtained as described above was packed in a Pyrex glass column (inner diameter Aw x length/θcrn), and
Aqueous solution containing θ sodium phosphate and 100 mM sodium chloride (pH 7 as mobile phase, room temperature, flow rate o, s)
Bovine trypsin o with d/via! When μm was injected, no protein with trypsin activity was eluted. Subsequently, the mobile phase was changed to 30mM +) sodium chloride, 100mM
When switching to an aqueous solution (...71 iL) containing M sodium chloride and 20 rr Mt-aminohexanoic acid, a component mainly composed of one protein with trypsin activity was eluted.

In addition, as a mobile phase liquid sending pump, KHU-2J 3A
(Kyowa Seimitsu ■), as a detector, R is used for protein detection.
F-330 (N Takasugi Seisakusho) Excitation wavelength, 2Ltnm.

Detection wavelength 3≠θnm, FD-/10 for enzyme activity detection
(JASCO Corporation ■) Excitation wavelength 3Ajnm, detection wavelength ≠A
Ovan was used. For detection of trypsin activity, 7-amizuge methyl, which is released by decomposition of the synthetic substrate t-butoxycarbonyl-L-glutamyl-L-lysyl-lysing-methylgumaryl-7-amide by trypsin, is used. The fluorescence of Kuq IJ y was utilized.

Example λ To the column packed in Example 1, 50mM! I Aqueous solution containing sodium chloride and 100mM sodium chloride (p
H7 Hiromu) as the mobile phase at room temperature and at a flow rate of 0. ! tnt/
Human plasminogen (green cross) with ygin 10
pt is high molecular weight urokinase (green cross)/jU
The activated mixture was injected for 3 minutes at 37° C. using Plasminogen, which does not have enzymatic activity, was only eluted in the transparent area, but then the mobile phase was
An aqueous solution (ff
When 174')K was switched, a protein with plasmin activity was eluted. Note that the same equipment, synthetic substrates, etc. as in Example 1 were used.

Example 3 After saponifying the granular copolymer obtained in Example 1, 10f of particles were placed in a beaker at 0θd, and 100yd of acetone, dried with molecular sieve≠A, was added.
-/'-carbonyldiimidazole 1AOj f was added and the reaction was carried out at room temperature for lj minutes with stirring to obtain an activated carrier having an imidazolyl carbamate group as an active group. t as a spacer molecule in the same manner as in Example 1.
-aminohexanoic acid is reacted with the activated carrier, t-
A spacer molecule-attached carrier on which aminohexanoic acid was immobilized was obtained. Immobilized t-aminohexanoic acid Jut/
/μmol/l spacer molecule-attached carrier.

In the same manner as in Example 1, p-aminobenzamidine was immobilized as a ligand molecule. 9. Calculate the amount of p-aminobenzamidine hydrochloride. ．． Everything was the same as in Example/, except that 2vati was used. The amount of immobilized p-aminobenzamidine was lrz μmol per 2 dry biological component-Cotter adsorption carriers.

The above adsorbent was placed in a Pyrex glass column (inner diameter t1
1IK x length/Octn), and using a water solution (pH 7≠) containing jOrrM sodium phosphate and 100mM sodium chloride as the mobile phase, at room temperature, flow rate o, rψ-
When bovine trypsin O1 μf was injected, no protein with trypsin activity was eluted. Subsequently, the mobile phase was switched to an aqueous solution (pH 1) containing OmM sodium phosphate, 100mM sodium chloride, and 20mM O-aminohexanoic acid, but no protein was eluted. Next, the mobile phase was changed to jOmM sodium phosphate, 1100rn
When switching to an aqueous solution containing sodium chloride, 20rnN (A-aminohexanoic acid and 3M urea (f z+),
A component consisting mainly of two proteins with trypsin activity was eluted. Note that the same equipment, synthetic substrates, etc. as in Examples were used.

Example G After saponifying the granular copolymer obtained in Example 1, particles 2.11 were placed in a 100 d beaker and dried with a molecular sieve 4IA of ace-30 = tons, 2! -1/, l'-carbonyldiimidazole,
lJ'4M was added to obtain an activated carrier having an imidazolyl carbamate group as an active group in the same manner as in Example 1, and a spacer molecule-attached carrier on which t-aminohexanoic acid was immobilized was obtained. The amount of immobilized 2-aminohexanoic acid was 3 t/μmol/f spacer molecule attached carrier.

In the same manner as in Example, p-aminopenzamidine was immobilized as a ligand molecule. The amount of p-aminopenzamidine hydrochloride was 2 g9. Everything was the same as in Example 1 except that /;s9 was used. The amount of immobilized p-aminopenzamidine was determined by
It was o1.

The above adsorbent was packed in a stainless steel column (inner diameter Aws x length 10 cm)'K, and an aqueous solution containing JOmM sodium phosphate and 1100rr sodium chloride (f
fl 74') as the mobile phase, room temperature, flow rate /, Og
Glu-plasminogen Zjμ2 and I in d/rm
ys-plasminogen 3. When a sample solution containing θμ2 was injected, Glu-plasminogen eluted later than the portion that passed through. Subsequently, the mobile phase was changed to jOrrM sodium phosphate, 100mM sodium chloride and 20t
Aqueous solution containing nM 6-aminohexanoic acid (pH 74')
When switched to , Lys-plasminogen was eluted.

Note that none of the detected proteins had plasmin activity. In addition, the device can also be used as a mobile phase pump.
The same material as in Example 1 was used except that INCLE (JASCO Corporation) was used.

Claims (4)

[Claims] (1) A method for separating trypsin-related enzymes by liquid chromatography, characterized in that a fully porous adsorbent having an alcoholic hydroxyl group and a ligand group having an affinity for the enzyme is used as a stationary phase. A method for isolating trypsin-related enzymes. (2) The fully porous adsorbent is made of a crosslinked copolymer, with 1.0 to 14.0 alcoholic hydroxyl groups per polymer weight.
mmol/g and ligand group 0.001-3000μm
o1/g and the amount of water that can be held is 0.5 to 4.0 g
2. The separation method according to claim 1, wherein the specific surface area is 5 to 1000 m^2/g. (3) The separation method according to claim 1 or 2, wherein the alcoholic hydroxyl group of the fully porous adsorbent is a hydroxyl group derived from a vinyl alcohol unit. (4) Claim 1, wherein the fully porous adsorbent has an amidino group or a guanidino group.
The separation method according to any one of Items 1 to 3.