JPH1099097A - Measurement of activity of enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring the activity of an enzyme, improved in specificity by using a synthetic substrate having the same partial structure as that of a natural substrate. SOLUTION: This method for measuring the activity of an enzyme comprises binding a substrate component having a chemical structure capable of being cleaved with an enzyme of measurement target and emitting fluorescent light also in a state not cleaved with the enzyme to a solid phase, and subsequently measuring fluorescent light depolarization factors before and after the breakage of the substrate component with the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring enzyme activity. More specifically, the present invention relates to qualitatively or quantitatively measuring the activity of the enzyme by measuring the degree of depolarization of a fluorescent dye released by cleavage by the enzyme.

[0002]

2. Description of the Related Art Many biological components have some physiological activity, and most of them are catalysts for chemical reactions and their inhibitory activities. Many chemical reactions involving biological components are hydrolyzed, and typical examples are enzymes and their inhibitors. Enzymes are responsible for much of the reactions in the living body, and the living body is maintained by antagonism with inhibitors. Typical examples of this scheme include blood coagulation and fibrinolytic systems.
Many proteases and their inhibitors are involved in the blood coagulation reaction, and measuring these factors is of great clinical importance.

Hitherto, the entire coagulation reaction has been measured using a coagulation time measuring method using plasma, but for each factor, a measuring method using a synthetic substrate or an immunological measurement has been performed. Among them, the synthetic substrate method utilizes a protease activity of a coagulation factor to detect a dye or a fluorescent substance released by the action of the factor. In other words, the active factor hydrolyzes a synthetic substrate as an enzyme in the same manner as a natural substrate, thereby releasing a dye or a fluorescent substance from the synthetic substrate. By detecting and quantifying this, the enzyme activity of the factor can be measured.

[0004] Conventionally used synthetic substrates include 1954
Year S. Synthesized TAMe such as Sherry (JBC, 20
8: 95-105, 1954) and several amino acids synthesized by Bromback et al. For example, Bz-Phe-Val-Arg-
pNA (Thromb. Research, 1: 267-278, 1972) and To
s-Gly-Pro-Arg-pNA (JP-A-52-3)
No. 492), HD-Phe-Pip-Arg-pNA
(JP-A-52-24590). These are used for the measurement of thrombin, and are subjected to an enzymatic reaction to release paranitroaniline (pNA), whereby a yellow color is obtained. By colorimetrically measuring the color at 405 nm, the coagulation / fibrinolytic ability is represented by the enzyme activity. be able to.

Also, Iwanaga et al. Have developed a fluorescent peptide substrate to which aminomethyl coumarin (AMS), which emits fluorescence when released, is bound (J. Biochem., 82: 1495-1498, 197).
7).

As described above, the activity of coagulation factors has been made measurable by using a synthetic substrate, but not all reagents are presently satisfactory. First, these synthetic substrates have no color or fluorescence in the substrate state, and are hydrolyzed by the enzyme to be measured to produce color or emit fluorescence only when the dye or fluorescent dye is released. This phenomenon does not occur in any case, but occurs when a specific substance is bonded to a specific site of a dye or a fluorescent dye. This is one of the factors that makes the development of new synthetic substrates difficult. The ability to bind to any substance broadens the use of synthetic substrates, but it is not easy.

[0007] The use of color development indicates that the specimen is also affected by coloring and turbidity.
The fluorescence is not completely unaffected by the sample.

[0008] The difficulty in developing new synthetic substrates is that
The point is that the cleavage portion of the enzyme must be between the peptide and the dye or fluorescent dye. As described above, these synthetic substrates have a structure that does not develop color when a dye is bound to a peptide. That indicates that the enzyme must act to release the dye itself. Since the original substrate of the enzyme is a protein or peptide, the cleavage part is between the peptides. However, cleavage between these synthetic substrates is between the peptide and the dye.

This means that it is necessary for the synthetic substrate to allow the enzyme to cut a place different from the natural site, which makes it difficult to produce the synthetic substrate. Is also a cause of low specificity.

[0010]

SUMMARY OF THE INVENTION To solve these problems, the present inventors have conducted intensive studies, and as a result, it has been found that an enzyme acts on a synthetic substrate having exactly the same structure as a natural substrate. Gives a larger signal change,
As a result, the inventors have found a measurement principle capable of measuring enzyme activity, and have completed the present invention.

[0011]

That is, the present invention has a chemical structure capable of being cleaved by an enzyme to be measured,
Further, the enzyme activity is characterized in that a substrate component that emits fluorescence even when the enzyme is not cleaved is bound to a solid phase, and the activity of the enzyme is measured by measuring the degree of depolarization of fluorescence before and after cleavage by the enzyme. Provide a measurement method.

In the present invention, when the substrate component is cleaved by an enzyme, the substrate component is separated into a first fragment that emits fluorescence and a second fragment containing the solid phase.

The principle of the measurement of the present invention will be described with reference to a method performed using a substrate component consisting of a peptide and a fluorescent dye.

A peptide containing an amino acid sequence that can be recognized and cleaved by an enzyme to be measured, and a substrate component consisting of a fluorescent dye bound to the tip thereof are constructed on a solid phase. The sample is reacted there. Then, the enzyme to be measured in the sample acts on the substrate component on the solid phase, cleaves the peptide at the specific recognition site, and emits a first fragment emitting fluorescence and a second fragment containing the solid phase.
Divided into fragments. At this time, the cleaved first fragment that emits fluorescence is in a free state. When bound to a solid phase, the motility of the fluorescent dye is small and the motility after release is high. Both can be easily distinguished by measuring the degree of depolarization of the fluorescent dye. Therefore, by measuring the degree of depolarization of the fluorescent dye before adding the sample and the degree of depolarization of the fluorescent dye after reacting the sample for a certain period of time, it is possible to know the activity of the enzyme to be measured in the sample. it can.

In the fluorescence depolarization measurement method, since the first fragment that emits fluorescence after cleavage is in a free state in a solution, the polarized light that is sent into the solution and excites the fluorescent dye is different between the incident light and the emitted light. This is based on the fact that depolarization occurs during light emission due to the rotation of fragments occurring between them. In this case, the degree of depolarization is inversely proportional to the turning rate of the fragment. In the state where the substrate component having the fluorescent dye at the tip is bound to the solid phase, the overall movement is extremely limited, and the polarized light is not depolarized from the solution until it is emitted from the time when the fluorescent dye is incident on the fluorescent dye. to go out.

The advantages of this method are that it uses a fluorescence depolarization detection method that is hardly affected by the sample and that the fluorescent dye must emit fluorescence both on a solid phase and in a free state. Can be placed in a portion away from the fluorescent dye. This allows the structure of the substrate component to be made so that the cleavage site is exactly the same as that of the natural substrate, and thus has higher substrate specificity than conventional synthetic substrates. In addition, it has become possible to produce substrate components for enzymes that could not be produced by conventional methods.

As the solid phase, any solid phase may be used as long as the degree of depolarization of the fluorescent dye is significantly different from that of the free dye. Commonly used microplates, test tubes, particles such as latex, and cells such as red blood cells can also be used.

Further, a water-soluble polymer can be used as the solid phase. In this case, it is necessary that there is a large difference between the degree of depolarization of the fluorescent dye in the state bound to the water-soluble polymer and the state in the free state. In other words, the substrate component is cleaved by the enzyme to separate into a first fragment that emits fluorescence and a second fragment containing a water-soluble polymer, and the second fragment has a molecular weight 100 times or more that of the first fragment. Preferably, it is large. Examples of the water-soluble polymer include dextran and polyethylene glycol. If there is a sufficient difference in molecular weight from the free state, proteins and the like can also be used as a solid phase.

The substrate component used in the present invention differs depending on the enzyme to be measured. If the enzyme to be measured is a protein or peptide degrading enzyme or its inhibitor,
The substrate component consists of a peptide containing a cleavage site and a fluorescent dye. If the enzyme to be measured is an enzyme that cleaves a sugar chain, the synthetic substrate consists of a sugar chain containing a cleavage site and a fluorescent dye.

As the enzyme which can be used in the present invention, any enzyme can be used as long as it has a strict substrate specificity. In particular, enzymes that are clinically highly significant include coagulation and fibrinolytic factors and inhibitors thereof. Since many of the coagulation factors are proteases or inhibitors thereof, the method of the present invention can be used to measure each factor. Factors currently measured by the synthetic substrate method include thrombin, factor Xa, plasma kallikrein, plasmin, urokinase, tissue kallikrein, etc., all of which are proteases, and these enzymes and The activity of the inhibitor can be measured.

The choice of the fluorescent dye is important because its fluorescence lifetime greatly affects the degree of fluorescence depolarization. For example, FITC (fluorescein isothiocyanate) and R
ITC (tetramethylrhodamine isothiocyanate)
By using such a method, a large signal can be obtained with a large quantum yield. In addition, these fluorescent dyes have a derivative that can be easily labeled to the peptide, and even if the derivative is labeled with the derivative, there is no significant change in the fluorescence characteristics, so that good measurement is possible. Particularly, FITC is preferable.

If a good difference in the degree of fluorescence depolarization cannot be obtained with FITC depending on the solid phase used, it is necessary to use another fluorescent dye having a different fluorescence lifetime. As a substance having a fluorescence lifetime longer than that of FITC, a rare earth complex can be exemplified. For example, Eu ³⁺ -(BCPDA) _n
(Wherein n is 2 or 3; BCPDA is 4,7-bis (chlorosulfonyl) -1,10-phenanthroline-2,9-dicarboxylic acid), or JP-A-2-88969, JP-A-1-
No. 127957, JP-A-64-47952, JP-T-Hei-6
No. 510296 can be used.

Such a substrate component can be prepared by a method known to those skilled in the art. For example, when the synthetic substrate is composed of a peptide and a fluorescent dye, the peptide chain can be synthesized by, for example, a solid phase synthesis method (Merrifield, J. Am. Chem. Soc. 85: 2149, 196).
3), can be synthesized by fragment condensation or solution synthesis. Most simply, it can be synthesized by a fully automatic synthesizer using a solid phase method. As the solid phase method, for example, the Fmoc method can be used. In this method, an amino acid protected with Fmoc is bound to a resin, followed by deprotection and washing of the Fmoc group, and subsequent condensation and deprotection of the Fmoc-protected amino acid.
By repeating the washing operation, peptide chains are sequentially synthesized.

At this time, the peptide chain is designed to include a cleavage site of the enzyme to be measured. The length of the peptide is not limited, but a length that shows sufficient specificity for the specific enzyme to be measured is required. If it is too short, it can be degraded by other enzymes that recognize similar structures. For example, a case where the enzyme to be measured is thrombin will be described. Thrombin breaks down fibrinogen to fibrin and fibrinopeptides A and B. Therefore, it is necessary to create a synthetic substrate that mimics the amino acid structure of the cleavage site of fibrinogen, which is a natural substrate. The amino acid structure and cleavage site of fibrinogen are as follows.

[0025]

Embedded image That is, there are two cleavage sites indicated by arrows. This time, the present inventor synthesized a substrate of the following structure close to the natural substrate: FITC-G1y-Pro-Arg-Val-Val-. It has the same structure as fibrinogen, which is degraded between Arg and Val.

The binding between the peptide chain and the fluorescent dye can be performed using a known method. The linkage may be by directly linking the two components or by way of a spacer. However, it is necessary for the fluorescent dye to bind so as not to interfere with the recognition function of the enzyme in the peptide, and therefore it is preferable to bind to one of both ends of the peptide chain.

Known methods can be used for binding the substrate component to the surface of the solid phase. What is necessary is just to couple the group of the solid phase surface to the group of the substrate component. When the substrate component contains a peptide, its amino group or carboxyl group can be used.

The binding between the water-soluble polymer and the substrate component can be performed in the same manner. The water-soluble polymer must have a functional group such as an amino group or a carboxyl group, and the functional group can be bonded to the C-terminal or N-terminal of the synthetic peptide using carbodiimide or glutaraldehyde. Alternatively, using various bivalent reagents,
Amino and thiol groups, carboxyl and thiol groups,
Although it is possible to bond a hydroxyl group to an amino group, a method using carbodiimide is considered to be the most common.

The degree of fluorescence extinction can be measured using a known device, for example, JIMCOMAC-II, IBF-
129 etc. can be used. FIG. 1 shows a schematic diagram of an apparatus for measuring the degree of fluorescence depolarization. When FITC is used as a fluorescent dye, the fluorescence excitation wavelength is 485 nm, the polarized light receiving wavelength is 525 nm, the spectral bandwidths of the wavelength filters on the excitation side and the light receiving side of the apparatus are both 10 nm, and the reaction cell is heated to 37 ° C. It is preferable to carry out the measurement while holding. To measure an enzymatic reaction using the method of the present invention,
Use a solution appropriate for the enzyme reaction to be measured. For example, when measuring thrombin, 15 mM imidazole,
0.135 M NaCl, 9 mM HCl, pH 7.4
And heparin buffer. Enzyme activity can be quantified as a decrease in fluorescence polarization (P) value over time.
In the following examples, the degree of depolarization of fluorescence is indicated as 1 / P.

The advantages of the measurement based on the degree of fluorescence depolarization are as follows: 1) The reaction mixture can be measured as it is without the need for precipitation or centrifugation, so that the operation is simple. 2) Radioimmunoassay or enzyme The measurement is quicker than the antibody method, and it can be measured in seconds to minutes.
3) Measurement is possible with sensitivity comparable to radioisotopes. 4) High stability of substrate components and high reproducibility after storage. 5) Safe because no radioactivity is used. 6) Short time. Since data is obtained, the same sample can be measured over time.

Further, in the method of the present invention, the fluorescent dye needs to emit fluorescence both on a solid phase and in a free state, so that the cleavage site of the enzyme can be placed at a position separated from the fluorescent dye. This allows the structure of the substrate component to be made so that the cleavage site is exactly the same as that of the natural substrate, and thus has higher substrate specificity than conventional synthetic substrates. In addition, it has become possible to produce substrate components for enzymes that could not be produced by conventional methods.

[0032]

EXAMPLES Example 1 Method for Measuring Thrombin Activity Preparation of Substrate Component A substrate component of the following structure: FITC-G1y-Pro-Arg-Val-Val- was prepared. The substrate is made by amino acid synthesis,
Synthesis was outsourced to an external organization. To bind to the solid phase,
There was a risk of steric hindrance.
Synthesis was performed with 5 residues of ys.

Binding of Substrate Component to Solid Phase Latex particles having a particle size of 0.75 μm were used as the solid phase. As the latex, polystyrene latex having an amino group on the surface (polybeads amino microspheres manufactured by Polyscience) was used.

The binding of the synthetic substrate to the latex was performed using water-soluble carbodiimide (EDC). The method followed the usual method.

1 m of 2.5% (w / v) latex particles
Add 100 μg of EDC per l. At this time, the solution contained 50 mM phosphate buffer and had a pH of 7. After reacting the solution at room temperature for 3 hours while stirring, 1
After performing centrifugation at 2000 rpm and removing the supernatant,
Add 1 ml of 10 μg / ml substrate component solution. After the reaction at room temperature for 3 hours, the same centrifugation treatment is performed and the supernatant is removed. The particles of the precipitate were dispersed in 0.1 M Tris buffer (pH 7), stored at 4 ° C. for 1 day, and then centrifuged again to remove 1% of BS.
A was dispersed in 50 mM Tris buffer containing A to obtain a substrate component binding latex.

The substrate component binding latex prepared as described above was measured for fluorescence intensity, and diluted to a concentration convenient for measurement.

Determination of thrombin activity The thrombin activity was measured using a kit of Test Team ATIII Auto ^™ of Daiichi Kagaku containing the following components: Thrombin agent: thrombin (from bovine) (quantity: 70 nk)
vial for at / 50ml) ・ Thrombin solution: sodium heparin (quantity: 100I)
U / 50ml vial) ・ Substrate: HD-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide dihydrochloride (S
-2238) (MW = 625.6) (quantity: 20 mg / 1
4 ml vial)-Substrate solution: surfactant The substrate was dissolved in the substrate solution, and the fluorescent substrate-bound latex was diluted 100-fold with the substrate solution.

The substrate component prepared this time was evaluated using the thrombin reagent of the above kit. A comparison was made using the synthetic substrate provided with this kit as a control.

140 μl of thrombin solution and 40 μl of substrate solution
After reacting at 37 ° C. for 5 minutes, absorbance at 415 nm (by the end point method) is measured in the case of the kit substrate, and the fluorescence depolarization of the fluorescent substrate-bound latex is performed in a reaction time of 5 minutes. The change in degree was measured.

The change in the degree of depolarization of fluorescence was measured using a self-made fluorescence depolarization measuring instrument. The thrombin solution was prepared by dissolving the thrombin agent included in the kit to a concentration 10 times with a thrombin solution, and measuring the change in absorbance and the degree of fluorescence depolarization when diluted twice with the same solution.

The results obtained are shown in Table 1 below. Table 1 Thrombin concentration (nkat / ml) Absorbance (415 nm) Depolarization of fluorescence 0 0.045 0.012 1.37 x 10 ^-2 0.095 0.230 2.73 x 10 ^-2 0.153 0.478 5.47 x 10 ^-2 0.272 0.952 1.09 x 10 ^-1 0.525 1.430 2.19 x 10 ^-1 0.964 1.583 4.38 x 10 ^-1 1.899 1.604 8.75 x 10 ^-1 3.046 1.611

In any of the methods, a change in signal was observed in accordance with the amount of thrombin added, confirming that thrombin could be measured. FIG. 2 shows the relationship between the thrombin activity and the absorbance, and the relationship between the thrombin activity and the relative fluorescence depolarization degree. It was shown that thrombin could be detected with higher sensitivity than conventional synthetic substrates.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a fluorescence depolarization degree measuring device used in the present invention.

FIG. 2 (A) shows the relationship between thrombin and absorbance when a control synthetic substrate is used, and (B) shows the relationship between thrombin and relative fluorescence depolarization when a substrate component of the present invention is used. Show.

Claims (4)

[Claims] 1. A fluorescent component which has a chemical structure capable of being cleaved by an enzyme to be measured and which emits fluorescence even in a state where the enzyme is not cleaved, to a solid phase, and before and after cleavage by the enzyme. A method for measuring enzyme activity, comprising measuring the activity of the enzyme by measuring the degree of dissolution. 2. The method according to claim 1, wherein the substrate component is separated into a first fragment that emits fluorescence when cleaved by an enzyme and a second fragment containing the solid phase. 3. The method according to claim 1, wherein the substrate component comprises a peptide and a fluorescent dye. 4. The solid phase is a water-soluble polymer, and when the substrate component is cleaved by an enzyme, the solid phase is divided into a first fragment that emits fluorescence and a second fragment containing the water-soluble polymer. The method of claim 1, wherein the second fragment has a molecular weight at least 100 times greater than the first fragment.