JPH0728756B2 - Enzyme activity measuring packing material, column packed with the same, and enzyme activity measuring method using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a packing material for measuring enzyme activity, an enzyme activity measuring column packed with the same, and a method for measuring enzyme activity using the same.

[Conventional technology]

Enzymes are proteins synthesized by living cells, and it is known that their catalytic action on biological reactions plays an important role in sustaining life. In the natural world, there are millions of enzymes of animal, plant or microbial origin, and some of the approximately 2,000 isolated among them are industrial enzymes that are related to foods and detergents. In addition, it has been widely used as a pharmaceutical enzyme for enzyme preparations, analysis, clinical tests, and in recent years for genetic engineering. Measuring the activity of an enzyme means measuring the catalytic ability of the enzyme, which is nothing but obtaining a basic index showing the function of the enzyme.
Conventionally, the measurement of enzyme activity has been performed by analyzing the amount of decrease in the substrate or the amount of increase in the product, and as a method therefor, an absorptiometer measures the amount of decrease in the substrate after the reaction depending on the type of enzyme. Often used are a method of measuring using a method, a method of measuring by coloring a substrate or a product with a chemical reagent, a method of converting the product into a coloring substance and measuring. There are various other methods such as labeling substrates with radioactive elements for detection, but generally speaking, the amount of enzyme to be measured is large, the reagents are expensive, the types of reagents used are many, and the measurement takes a long time. However, there are drawbacks such as requiring skill in measurement technique, being easily affected by impurities, and being difficult to obtain an accurate value. As one of the measures against these drawbacks, separation and purification of the enzyme by a column is performed by combining with liquid chromatography, and after the column is eluted, it is contacted with a substrate solution to measure the reduction amount of the substrate. [Etch. A. Chase: Journal of Chemical Technology and Biotechnology, Vol. 36, 351 pp., 1986 (HAChase: J.Chem.Tech.Biotechnol, 36, 351 (198
6)) and Naofumi Ito et al .: Jonal of Chromatography, 400, 163, 1987 (N.Ito et al; J.Chrom
atogr. 400 , 163 (1987))]. However, this method is not always satisfactory in that an expensive reagent needs to be continuously flown and a new device such as a pump is required for that purpose.

[Problems to be Solved by the Invention]

An object of the present invention is to overcome the drawbacks of the conventional methods for measuring enzyme activity, to enable convenient, rapid, and highly accurate measurement of enzyme activity, a packing material for measuring enzyme activity, a column packed with the same, and the same. Another object of the present invention is to provide a method for measuring the enzyme activity using.

[Means for Solving the Problems]

The present invention provides a packing material for measuring enzyme activity, a column packed with the packing material, and a method for measuring enzyme activity using the packing material, which can achieve the above object.

That is, in the present invention, (a) an eluent is sent by a high-pressure pump, (b) a sample solution containing an enzyme is injected into the flow of the eluent, and (c) an eluent in which the sample solution is injected. Through a column packed with a packing material in which the substrate is immobilized to decompose the substrate by the reaction between the enzyme and the substrate,
(D) The packing material used in a flow-type enzyme activity measuring method, which comprises quantifying a substrate decomposition product in a liquid flowing out from the column, which is in contact with an enzyme in an eluent containing a sample liquid Substrate that produces a water-soluble substrate decomposition product that can be detected by means of a bonding group having a length of 6 to 30 atoms and a particle size of 3 to 100 μm.
The present invention relates to a packing material for enzyme activity measurement, characterized in that it is immobilized on a hard bead support of m by covalent bond.

In the present invention, (a) an eluent is sent by a high-pressure pump, (b) a sample solution containing an enzyme is injected into the flow of the eluent, and (c) an eluent in which the sample solution is injected. Through a column packed with a packing material in which the substrate is immobilized,
A column used in a flow-type enzyme activity measuring method, which comprises decomposing a substrate by a reaction between an enzyme and a substrate, and (d) quantifying a substrate decomposition product in a liquid flowing out from the column, comprising: The substrate, which produces a water-soluble substrate decomposition product that can be detected by contact with an enzyme in an eluent containing a liquid, has a particle size of 3 to 100 μm via a bonding group having a length of 6 to 30 atoms.
The present invention relates to a column for measuring enzyme activity, which comprises a hard bead support of m and a packing material which is immobilized by covalent bonding.

Furthermore, in the present invention, (a) the eluent is sent by a high-pressure pump, (b) the sample solution containing the enzyme is injected into the flow of the eluent, and (c) the enzyme solution in the sample solution is contacted. A substrate that yields a detectable water-soluble substrate degradation product by
Through the bonding group having the length of 6 to 30 atoms, the particle size of 3 to
A column decomposition product is produced by passing an eluent in which the above sample solution is injected into a column filled with a packing material which is immobilized by covalent bonding on a hard bead support of 100 μm, and (d) flows out from the column. Flow-type enzyme activity measuring method characterized by quantifying a substrate decomposition product in the liquid; and (a) sending an eluent by a high-pressure pump, and (b) a sample liquid containing the enzyme in the eluent. (C) Water solubility detectable by contacting with the enzyme in the sample solution, (c) the eluent in which the sample solution is injected is passed through a separation column to separate the enzyme in the solution, and Substrate producing a decomposition product of a substrate was filled with a packing material, which was covalently immobilized on a rigid bead support having a particle size of 3 to 100 μm through a bonding group having a length of 6 to 30 atoms. Flow through the column from the separation column above. The present invention relates to a flow-type enzyme activity measuring method, characterized in that a substrate decomposition product is produced through the discharged liquid, and (e) the substrate decomposition product in the liquid flowing out from the column is quantified.

Hereinafter, the packing material for measuring enzyme activity of the present invention, the column packed with the packing material, and the method for measuring enzyme activity using the packing material will be described.

The packing material for measuring enzyme activity of the present invention is prepared by covalently immobilizing a substrate recognized by an enzyme on a support made of water-insoluble hard beads.

The water-insoluble support used may be either porous or non-porous, but silica gel and hard beads of synthetic polymer such as styrene polymer, vinyl alcohol polymer and methacrylate polymer are used. . Cellulose, which has been often used in the past,
Polysaccharides such as agarose, dextran and so-called soft beads such as polyacrylamide are not suitable. As the hard beads, those having a particle diameter in the range of 3 to 100 μm and as constant as possible are used. If the particle size of the hard beads is less than 3 μm, it is difficult to increase the flow rate due to excessive pressure applied to the solution, and it is also difficult to make the particle size uniform. On the contrary, if the particle size exceeds 100 μm, fine packing cannot be performed, and it becomes difficult to obtain a sufficient surface area for immobilizing the substrate. In either case, it becomes difficult to repeatedly perform highly accurate measurement in a short time.

In order to bind the substrate recognized by the enzyme to the support, it is first necessary to attach the binding group-introducing group of the support, for example, the binding group having a functional group capable of covalently binding the substrate to the hydroxyl group.

As the functional group, an epoxy group, an amino group, a hydrazino group,
Examples thereof include a carboxyl group and a formyl group. The introduction of the binding group having these functional groups into the support can be easily carried out by a known method in the presence of a suitable solvent. Examples of the bonding group having an epoxy group include epihalohydrins such as epichlorohydrin, diglycidyl ethers such as 1,4-butanediol diglycidyl ether, and epoxysides such as 1,7-octadiene diepoxide. Etc., but these react with the hydroxyl groups on the support immediately under alkaline conditions to give an epoxy-modified support. The obtained epoxy-modified support is reacted with ammonia, hydrazine, or a diaminoalkane such as 1,3-propanediamine to give an amino-modified support having an amino group, and a 4-aminobutyric acid support. A carboxyl-modified support is obtained by reacting with such an aminocarboxylic acid, and a formyl-modified support is obtained by hydrolyzing an epoxy group and performing periodate oxidation. The amino-modified support can also be converted to a carboxyl-modified support by reacting it with an acid anhydride such as succinic anhydride. When binding a substrate recognized by an enzyme to a support having these functional groups, if necessary depending on the functional group of the binding group, it is carried out in an appropriate solvent using an appropriate catalyst or reaction reagent. be able to. For example, as a catalyst, an acid such as hydrochloric acid or sodium carbonate or sodium hydrogen carbonate, or an alkali is mainly used when the functional group is an epoxy group, and as a reaction reagent, for example, N-hydroxysuccinimide, dicyclohexylcarbodiimide or 1-ethyl-3-
Illustrate that a condensing agent such as (3-dimethylaminopropyl) carbodiimide is used when the functional group is a carboxyl group, a reducing agent such as sodium cyanoborohydride is used when the functional group is a formyl group, etc. You can Water is usually used as the solvent. If necessary, it may be used as a phosphoric acid or acetic acid buffer solution, or an inorganic salt such as sodium chloride may be added and used.

As the linking group, one having a length of 6 to 30 is used. If the length of the binding group is too short, the substrate cannot bind to the binding site of the enzyme due to steric hindrance of the enzyme itself, and the desired enzymatic reaction cannot be achieved. On the contrary, when the length of the bonding group exceeds 30 atoms, the steric freedom of the substrate bonded to the end of the bonding group is increased, and the physicochemical interaction on the surface of the hard bead is strengthened. However, it becomes difficult to bind the substrate.

The substrate recognized by the enzyme immobilized on the support having a functional group is not particularly limited as long as it produces a substrate decomposition product upon contact with the corresponding enzyme whose activity is to be measured. Typical examples of the substrate include amines such as glucose, cholesterol, choline, uric acid, benzylamine and histamine, xanthine, acetylcholine, glutathione, thymidine-5'-phosphate, chondroitin sulfate,
Phosphoric acid monoesters such as (deoxy) ribonucleic acid, cholesterol ester, bisparanitrophenyl phosphate,
Amino acids such as arginine, lysine, and tyrosine, phenylalanylseryl arginine, which has a C-terminal-specific amino acid, and a labeling compound such as paranitrophenyl group bound to the C-terminus, such as glycylglutamic acid Ester linkage of various oligopeptides, synthetic substrates such as benzoylarginine ethyl ester, α- (β-) glucose, α- (β-)
Galactose, α- (β-) mannose, β-fructose, α, α-trehalose, β-glucuronic acid, α-
Those in which L-fucose is glycoside-linked with another sugar such as glucose or with a labeling compound such as methylumbelliferone, cellulose, chitin, dextran, starch, heparin, fructose-1,6-bisphosphate, etc. can give.

The enzyme activity-determining filler obtained by immobilizing the substrate recognized by the enzyme obtained as described above on a water-insoluble support is usually filled in a hollow tube according to a conventional method,
Used as a column for measuring enzyme activity.

The material of the hollow tube filled with the enzyme activity measuring filler is generally glass, stainless steel, Teflon, stainless steel whose inner wall is coated with Teflon, or the like. The size of the hollow tube may be appropriately determined according to the purpose and is not particularly limited.

The method for filling the hollow tube with the enzyme activity-measuring filler is a conventional method and is not particularly limited. Further, the filling rate may be appropriately selected.

To measure the enzyme activity by high performance liquid chromatography using the enzyme activity measuring column obtained by the present invention, the eluent is sent by a high-pressure pump, and the sample solution containing the enzyme is introduced into the flow of the eluent. After injecting, and if necessary, the eluent in which the sample solution has been injected is passed through a separation column and then through a column packed with a packing material in which the substrate is immobilized, and the substrate is reacted by the enzyme and the substrate. It is carried out by decomposing and quantifying a substrate decomposition product generated by contact between the enzyme and the substrate. That is, when an enzyme to be measured for activity is injected into a column for measuring enzyme activity, as the enzyme to be measured for activity passes through the column, an amount of a substrate degradation product corresponding to the activity of the enzyme of interest is generated, and the generated substrate is decomposed. An object can be detected by using a detector such as ultraviolet absorption, fluorescence absorption, refractive index, or an electrochemical detector. The measurement temperature is
There is no particular limitation, but a range of 4 to 60 ° C is usually desirable.
The enzyme whose activity is to be measured is not particularly limited, and all enzymes can be applied. The enzyme whose activity is to be measured may be a purified single preparation, a crudely purified preparation, or a single component present in a biological sample. Especially when other substances such as other contaminating proteins are present in the sample, combine with a separation column such as gel permeation, ion exchange, or hydrophobic chromatography, and use the enzyme activity measurement column as a post column. be able to. In such a post-column method, it is possible to immediately know at which portion of the peak separated by the separation gel the activity is present at the same time as measuring the enzyme activity. By incorporating it in a high performance liquid chromatography device, it is possible to measure the enzyme activity with high sensitivity even in a small amount of sample in a short time, and it is also advantageous to combine it with various other separation columns.

The substrate immobilized on the support used in the present invention may be any one that produces a water-soluble substrate decomposition product upon contact with an enzyme. For example, when oxidase, hydrolase, lyase, etc. are applied as the enzyme whose activity is to be measured,
Preferable combinations of these enzymes whose activity is to be measured and the substrates and methods for detecting the degradation products of the substrates generated from the system combining them will be exemplified.

First, as substrates corresponding to oxidases such as glucose oxidase, cholesterol oxidase, choline oxidase, urate oxidase, amine oxidase, and xanthine oxidase, glucose, cholesterol, choline, uric acid, amines such as benzylamine and histamine, respectively, And xanthine. Substrate decomposition products generated in these systems are mainly hydrogen peroxide, and can be easily detected and measured electrochemically, for example.

The hydrolases include esterase, protease, glycosidase and the like. The esterase may be of any type, but includes acetylcholinesterase, glutathione thiol ester hydrolase, phosphodiesterase I, chondroitin sulfatase,
In nucleases such as (deoxy) ribonucleases I and II, cholesterol esterase, alkaline phosphatase, etc., acetylcholine, glutathione, thymidine-5'-phosphate,
Mention may be made of phosphoric acid monoesters such as chondroitin sulfate, (deoxy) ribonucleic acid, cholesterol ester and bisparanitrophenyl phosphate. Substrate degradation products produced in these systems are cholingurtathion, thymidine, chondroitin, nucleotides, cholesterol,
Para-nitrophenol and the like, all of which can be detected and measured by ultraviolet absorption, fluorescence absorption, or change in refractive index.

In proteases such as trypsin, plasmin, urokinase, bromelain, pronase, chymopapain pepsin, and chymotrypsin, the substrates include arginine, lysine, tyrosine, etc. As described above, a compound having a labeling compound such as a paranitrophenyl group bound to the C-terminus of an oligopeptide having a specific amino acid at the C-terminus (hereinafter referred to as (A)), or an oligopeptide such as glycylglutamic acid is ester-bonded (Hereinafter referred to as (B)) or a synthetic substrate such as benzoylarginine ethyl ester (hereinafter referred to as (C)). In the case of these proteases, labeled compounds or oligopeptides bound to specific amino acids are produced in the above systems (A) and (B), and decomposition products of synthetic substrates are produced in the system (C). come. Therefore, detection of these products can be appropriately selected depending on the properties of the produced compound. When the product is a labeled compound, compounds having ultraviolet absorption such as para-nitrophenol and para-nitroaniline are detected and measured by an ultraviolet spectrophotometer. Further, in the case of a compound having fluorescence absorption such as 4-aminomethylcoumarin, it is detected and measured by a fluorometer. When a synthetic substrate decomposition product having an oligopeptide and a benzoyl group or a dansyl group is produced, it can be detected and measured using an ultraviolet spectrophotometer or a refractometer.

Among glycosidases, α- (β-) glucosidase, α- (β-) galactosidase, α- (β-) mannosidase, β-fructosidase, α, α-trehalase, β-glucuronidase, α- (L-). There are fucosidases and the like, and the respective substrates are α- (β-) glucose, α- (β-) galactose, α- (β-) mannose, β-fructose, α, α-trehalose, β
-Glucuronic acid and α-L-fucose may be glycosidically linked to other sugars such as glucose or to a labeling compound such as methylumvariferone. Substrates for glycosidases having specificity for polysaccharides such as cellulase, chitinase, dextranase, diastase, and heparinase include cellulose, chitin, dextran, starch and heparin.

In these glycosidases, when the degradation product is sugar, it can be detected and measured by a refractometer, and when it is a labeled compound having ultraviolet absorption or fluorescence absorption, it can be detected and measured by an ultraviolet spectrophotometer or a fluorometer. it can.

Further, examples of the lyase include aldolases such as fructose bisphosphate aldolase.
As the substrate, use fructose-1,6-bisphosphoric acid, etc. to detect and measure the decomposition products such as dihydroxyacetone phosphoric acid or glyceraldehyde-3-phosphoric acid using an ultraviolet spectrophotometer or differential refractometer. You can

〔Example〕

Hereinafter, the present invention will be described more specifically by showing typical examples. Needless to say, these are merely examples for explanation and the present invention is not limited to these.

Example 1 Hydroxyl group-modified beads obtained from glycidyl methacrylate and ethylene glycol dimethacrylate were mixed with 1M NaOH.
Then, 1,4-butanediol diglycidyl ether was reacted to introduce an epoxy group, and then a carboxyl group was introduced with γ-aminobutyric acid. The obtained beads were thoroughly washed with anhydrous dioxane to give carboxyl-modified beads. N-Hydroxysuccinimide and dicyclohexylcarbodiimide were added to the carboxyl-modified beads in anhydrous dioxane, the mixture was reacted by shaking at room temperature for 1.5 hours, and then the gel was taken and washed quickly with anhydrous dioxane and methanol. 1 g of this gel was added to 30 mg of arginine paranitroanilide (Agr-pNA) in 0.01N carbonate buffer (pH 9.4) water.
The mixture was added to 3 ml, shaken at room temperature for 2 hours, and left at 4 ° C. overnight. The gel was then removed and washed with 1M aqueous sodium chloride solution and water. It was confirmed that the Agr-pNA-immobilized gel thus obtained carried about 10 μmolo of Agr-pNA per 1 g of dried gel.

The obtained Agr-pNA-immobilized beads had an inner diameter of 4.6 mm and a length of 1.0.
A cm column made of stainless steel was packed to obtain an Agr-pNA immobilized column. An Agr-pNA-immobilized column was incorporated into a high-performance liquid chromatograph (Fig. 1), trypsin activity was measured, and the detection limit, linearity, and flow rate dependence of the measurement in this device were examined.

(1) Detection limit of trypsin activity in this device and linearity of the measured value As the trypsin activity value injected into this device increased, the measured value detected increased linearly, showing a good correlation (Fig. 2). ). The detection limit was 0.05 nkat. The chromatographic conditions are as shown below.

Eluent: 50 mM Tris-HCl buffer pH7.4 + 0.15M NaCl
(Eluent A) Flow rate: 1 ml / min Detection: 405 nm Sample: Trypsin (Sigma) BAEE (benzoylarginine ethyl ester) nkat / 3μ Eluent A + 0.5 mM
CaCl ₂ However, the activity of trypsin is shown by the hydrolysis activity against BAEE, and 1BAEE nkat is defined as the amount of enzyme activity that decomposes 1 nmolo BAEE in 1 second.

Measurement temperature: 25 ° C (2) Flow rate dependence of measurement values A fixed amount of trypsin was injected into this device, and the dependence of measurement values on flow rate is shown in Fig. 3.

Chromatography conditions are in accordance with (1) except for the flow rate.

As a result, as shown in FIG. 3, the value tended to decrease in inverse proportion to the increase in flow velocity.

(3) Temperature Dependence of Measured Value A certain amount of trypsin was injected into this device, and the dependency of measured value on temperature is shown in FIG. Chromatography conditions are in accordance with (1) except temperature. As a result, as shown in FIG. 4, it was found that the measured value reached the maximum around 40 ° C. of the column temperature.

(4) Dependence of measured values on salt concentration A certain amount of trypsin was injected into this device, and the dependency of measured values on salt concentration is shown in FIG. Chromatography conditions are in accordance with (1) except for salt concentration.

As a result, as shown in FIG. 5, the measured value tended to decrease as the salt concentration increased.

Example 2 The Agr-pNA-immobilized beads obtained in Example 1 were treated with an inner diameter of 4.6 m.
Agr-pNA was packed in a stainless steel column with a length of 1.0 cm and a length of 1.0 cm.
The column was immobilized. Also, a commercially available gel overseparation column (WS-803, Showa Denko KK) with an inner diameter of 8.0 mm and a length of 30 cm
This was connected to form a separation column. A separation column and an Agr-pNA immobilized column were installed in a high performance liquid chromatograph (FIG. 6), and a mixture of four kinds of proteins (thyroglobulin, transferrin, ovalbumin, trypsin) was injected into the device. 28 protein separation patterns
0 nm, or trypsin activity was detected by absorption at 405 nm. As a result, as shown in FIG. 7, it was found that this apparatus can simultaneously separate several proteins (FIG. 7-1) and detect trypsin activity (FIG. 7-2).

The chromatographic conditions are as shown below.

Eluent: 50 mM Tris-HCl buffer pH7.4 + 0.15M NaCl Flow rate: 1 ml / min Detection: 405 nm Sample: Tyroglobulin (bovine, type I, Sigma) Transferrin (human, Sigma) Ovalbumin (Manufactured by Sigma) Trypsin (bovine pancreas, type III) Measurement temperature: 25 ° C. Example 3 D-phenylpipecoyl arginine paranitroanilide was used instead of arginine paranitroanilide in Example 1, and trypsin was used instead. When thrombin was used and the linearity of the measured thrombin activity value in this device was examined according to Example 1 except for the above, the measured value detected was linear with the increase in the thrombin activity value injected into this device. It rose and showed a good correlation (FIG. 8). In addition,
Chromatographic conditions are as shown below.

Eluent: 50mM Tris-HCl buffer pH7.4 + 0.15M NaCl Flow rate: 1ml / min Detection: 405nm Sample: Thrombin ((Sigma, human plasma) NIH uni
t / 5μ 50 mM sodium citrate (pH 0.65) + 0.15M
NaCl measurement temperature: 25 ° C. [Effect of the invention] The enzyme activity measuring method according to the present invention is a simple, rapid and highly accurate method that overcomes the drawbacks of the conventional enzyme activity measuring methods. It provides a useful tool in various fields related to.

[Brief description of drawings]

FIG. 1 is a schematic view of an enzyme activity measuring device according to the present invention. However, in the figure, a pump, a trypsin, a thermostat, an Agr-pNA immobilized column, and a detector. FIG. 2 shows the correlation between the activity value of injected trypsin and the measured value obtained. In the figure, the vertical axis represents the peak area of pNA (paranitroaniline) which is a decomposition product (× 10 ^-3
= PNA μmole), the horizontal axis represents the activity of injected trypsin (B
AEEnkat / 3μ) is shown. FIG. 3 shows the relationship between the flow velocity and the measured value. In the figure, the vertical axis represents the peak area of pNA (× 10 ⁻⁴ = pNA μmole), and the horizontal axis represents the flow rate (ml / min). FIG. 4 shows the relationship between the temperature and the measured value. In the figure, the vertical axis represents the peak area of pNA (× 10 ^-4 = pNA μmole),
The horizontal axis represents temperature (° C). FIG. 5 shows the relationship between the concentration of sodium chloride contained in the eluent and the measured value. In the figure, the vertical axis represents the peak area of pNA (× 10 ^-4 = pNA μmole), and the horizontal axis represents the molar concentration (M).
Indicates. FIG. 6 is a schematic diagram of an enzyme activity measuring device when a separation column is used in combination with a substrate column. However, in the figure,
Is a pump, is a sample, is a constant temperature bath, is a separation column,
Indicates an Agr-pNA immobilized column, and indicates a detector. FIG. 7 is a chromatogram when the separation column is used in combination with the substrate column. Figure 7-1 shows the protein separation pattern (by absorption at 280 nm) and Figure 7-
2 shows the activity of trypsin (peak of pNA due to absorption at 405 nm). However, in the figure, indicates a thyroglobulin peak, a transferrin peak, an ovalbumin peak, and a trypsin peak. FIG. 8 shows the correlation between the activity value of the injected thrombin and the obtained measurement value. In the figure, the vertical axis represents the peak area of pNA, and the horizontal axis represents the activity of injected thrombin (NIH un
it).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP 62-265998 (JP, A) JP 57-18996 (JP, A) JP 60-30698 (JP, A) JP 60- 235058 (JP, A) Actually opened Sho 60-183555 (JP, U)

Claims (4)

[Claims] 1. An eluent is sent by a high-pressure pump, (b) a sample solution containing an enzyme is injected into a stream of the eluent, and (c) an eluent in which the sample solution is injected. Passing through a column packed with a packing material in which the substrate is immobilized to decompose the substrate by the reaction between the enzyme and the substrate, and (d) quantifying the substrate decomposition product in the liquid flowing out from the column. In the above-mentioned packing material used in the flow-type enzyme activity measuring method, the substrate which produces a detectable water-soluble substrate decomposition product upon contact with an enzyme in an eluent containing a sample solution has 6 atoms. Through a linking group having a length of ~ 30, a particle size of 3 ~ 100μ
A packing material for enzyme activity measurement, which is immobilized on a hard bead support of m by covalent bond. 2. An (a) eluent is sent by a high-pressure pump, (b) a sample solution containing an enzyme is injected into the flow of the eluent, and (c) an eluent in which the sample solution is injected. Passing through a column packed with a packing material in which the substrate is immobilized to decompose the substrate by the reaction between the enzyme and the substrate, and (d) quantifying the substrate decomposition product in the liquid flowing out from the column. In the above-mentioned column used in the flow-type enzyme activity measuring method comprising, the substrate producing a water-soluble substrate decomposition product that can be detected by contact with an enzyme in an eluent containing a sample solution has 6 to 6 atoms. Through the linking group having a length of 30, the particle size of 3 ~ 100μ
A column for measuring enzyme activity, characterized in that it is filled with a packing material that is immobilized by covalent bonding on a hard bead support of m. 3. A method comprising: (a) sending an eluent by a high-pressure pump, (b) injecting a sample solution containing an enzyme into a stream of the eluent, and (c) contacting the enzyme in the sample solution. The substrate that produces a water-soluble substrate decomposition product that can be detected by means of a linking group having a length of 6 to 30 atoms has a particle size of 3 to 100.
A column-decomposed product is produced by passing an eluent in which the above-mentioned sample solution is injected into a column filled with a packing material which is immobilized by covalent bonding on a hard bead support of μm,
(D) A flow-type enzyme activity measuring method, which comprises quantifying a substrate decomposition product in a liquid flowing out from the column. 4. An eluent is sent by a high-pressure pump, (b) a sample solution containing an enzyme is injected into the flow of the eluent, and (c) an eluent in which the sample solution is injected. The substrate which separates the enzyme in the liquid through the separation column and produces a detectable water-soluble substrate decomposition product by (d) contacting with the enzyme in the sample liquid has a length of 6 to 30 atoms. A liquid that flows out from the separation column is passed through a column packed with a packing which is immobilized by covalent bonding on a hard bead support having a particle size of 3 to 100 μm via a binding group to generate a substrate decomposition product. (E) A flow-type enzyme activity measuring method, which comprises quantifying a substrate decomposition product in a liquid flowing out from the column.