JP4797349B2 - Method of stabilizing reagent using vitamin B6 enzyme and reagent - Google Patents

Description

  The present invention relates to a technique for stabilizing a reagent using a vitamin B6 enzyme.

The vitamin B6 enzyme is an enzyme involved in the entire amino acid metabolism such as deamination, decarboxylation, racemization, and aldol cleavage. For example, this enzyme uses pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP) as a coenzyme. An example of a reagent using vitamin B6 enzyme is a tyrosine measuring reagent. This reagent uses tyrosine decarboxylase, which is a vitamin B6 enzyme, converts tyrosine in serum to tyramine, acts tyramine oxidase on tyramine to produce hydrogen peroxide, and hydrogen peroxide is added in the presence of peroxidase. It is for determining the tyrosine concentration by oxidizing and condensing 4-aminoantipyrine and measuring the absorbance (see, for example, Patent Document 1). In the medical field, the ratio of total branched chain amino acid to tyrosine concentration is used to determine the severity of liver disease and as a marker for diagnosing transition from chronic hepatitis to cirrhosis. The tyrosine concentration in serum is about several tens of μmol / L in healthy subjects, and measurement requires accuracy.
JP 63-226299 A

  An object of the present invention is to provide a stabilizing method and a stabilizing reagent that stabilize the activity expression of the vitamin B6 enzyme and improve the stability and precision of a reagent using the enzyme, for example, a tyrosine measuring reagent.

The present invention relates to the following reagents and stabilization methods.
1. A reagent having at least two parts including a vitamin B6 enzyme and a coenzyme of the vitamin B6 enzyme, wherein the vitamin B6 enzyme and the coenzyme of the vitamin B6 enzyme are formulated into separate parts.
2. Item 5. The reagent according to Item 1, wherein the vitamin B6 enzyme is tyrosine decarboxylase.
3. Item 2. The reagent according to Item 1, wherein the coenzyme is pyridoxal 5-phosphate.
4). The reagent according to any one of claims 1 to 3, wherein the reagent using vitamin B6 enzyme is a tyrosine measuring reagent.
5. A method and reagent for stabilizing vitamin B6 enzyme coenzyme in a reagent containing vitamin B6 enzyme and a coenzyme of the enzyme, wherein the vitamin B6 enzyme and the coenzyme of vitamin B6 enzyme are separately formulated as a composition during storage A method and a reagent characterized in that
6). Item 6. The method and reagent according to Item 5, wherein the vitamin B6 enzyme is tyrosine decarboxylase.
7). Item 6. The method and reagent according to Item 5, wherein the coenzyme is pyridoxal 5-phosphate.
8). Item 8. The method and reagent according to any one of Items 5 to 7, wherein the reagent using vitamin B6 enzyme is a tyrosine measuring reagent.

  According to the present invention, the activity expression of the vitamin B6 enzyme to which a coenzyme is bound can be stabilized, and the stability and precision of a reagent using the enzyme, for example, a tyrosine measurement reagent can be improved.

  The vitamin B6 enzyme used in the present invention is not particularly limited as long as it is an enzyme having pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP) as a coenzyme. For example, amino acid decarboxylase, amino acid Examples include enzymes related to amino acid metabolism such as racemase and transaminase, and muscle phosphorylase. More specifically, tyrosine decarboxylase, aspartate aminotransferase and the like can be mentioned.

  As the vitamin B6 enzyme of the present invention, an apo-enzyme obtained by removing a coenzyme by desalting or the like, or a holo-enzyme bound with a coenzyme may be used. When 100% coenzyme is bound to vitamin B6 enzyme (fully holoenzyme), it is not necessary to add additional coenzyme. Since apo-enzymes may be mixed for storage reasons, such incomplete holo-enzymes are also formulated separately from the coenzyme in the present invention to stabilize the activity expression. Included in the possible vitamin B6 enzyme.

  The reagent of the present invention may be in a liquid state such as an aqueous solution or a solid state such as lyophilized or powdered, but is generally in a liquid state (liquid reagent or The effect is more exhibited in maintaining stability after dissolving the freeze-dried product).

  The storage temperature is refrigerated conditions, that is, a temperature not exceeding 10 ° C. and freezing, preferably around 4 ° C.

  In a preferred embodiment of the present invention, the vitamin B6 enzyme and the coenzyme of the vitamin B6 enzyme are each formulated separately as a solution, and are used together in use. When a vitamin B6 enzyme and a coenzyme of the vitamin B6 enzyme are formulated as a solution, the pH is usually about 5 to 10, preferably about pH 6 to 8, more preferably about pH 6 to 7.5. The solution is preferably a buffer. As the buffer solution, phosphate buffer solution, Tris-HCl buffer solution and the like are preferable.

  The stabilization mechanism of the present invention is to prevent coenzyme degradation, which is a destabilizing factor, from occurring in the presence of an enzyme. The degradation of the coenzyme is considered to be mainly due to oxidation by light, but the coenzyme degradation factor in the present invention is not particularly limited.

Although not wishing to be bound by theory, the present inventor has the following reason why the activity expression of the vitamin B6 enzyme can be stabilized by separately formulating the vitamin B6 enzyme and its coenzyme. I guess so.
Vitamin B6 enzyme (apo-type enzyme) that is not bound to coenzyme and its coenzyme coexist in one part, vitamin B6 enzyme (apo-type enzyme) and its coenzyme are combined during storage and holo-type enzyme become.

  When the coenzyme deteriorates in the holo-type enzyme, the activity expression of the vitamin B6 enzyme remains lowered even if a new coenzyme not deteriorated is added. This is presumably because the replacement of the degraded coenzyme with the vitamin B6 enzyme does not occur easily.

Even when vitamin B6 enzyme (apo-type enzyme) and its coenzyme are formulated separately (for example, in separate parts), some degradation of the coenzyme can occur in the same way as the holo-type enzyme,
When a vitamin B6 enzyme (apo-type enzyme) and its coenzyme are mixed immediately before use, the activity expression of the vitamin B6 enzyme is not reduced, and the activity expression of the enzyme is stabilized. One interpretation of this phenomenon is that the present inventors selectively bind vitamin B6 enzyme (apo-type enzyme) to an undegraded coenzyme rather than a degraded coenzyme. Even if it deteriorates, it is considered that the activity of vitamin B6 enzyme (holo-type enzyme) does not decrease.

  The present invention is particularly advantageous when preparing a tyrosine measurement reagent.

  Measurement of tyrosine using a tyrosine measuring reagent can be performed, for example, as follows:

* EHSPT: N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine Specifically, when tyrosine decarboxylase is allowed to act on tyrosine in a sample, it is converted to tyramine and CO2, and then tyramine oxidase Is decomposed into 4-hydroxyphenylacetaldehyde, NH ₃ and H ₂ O ₂ . The produced H ₂ O ₂ can be oxidized and condensed with 4-aminoantipyrine and EHSPT under the action of peroxidase, the absorbance of the resulting quinone dye can be measured, and the amount of tyrosine can be measured.

In one particularly preferred embodiment of the present invention, the tyrosine measurement reagent comprises the following:
Vitamin B6 enzyme (peroxidase, tyramine oxidase as the first reagent, tyramine decarboxylase as the second reagent);
・ Coenzyme (vitamin B6)
A dye system that is visualized by reaction with hydrogen peroxide generated in the presence of peroxidase (eg, N-ethyl (2-hydroxy-3-sulfopropyl) -m-toluidine as the first reagent, 4- Aminoantipyrine); and buffering agents.

It is desirable to divide the enzyme into the first reagent and the second reagent so that the enzyme can be measured by an automatic analyzer (for example, Hitachi 7170 type automatic analyzer). In this way, the storage stability of the reagent is further improved. Can be made. Also, the coenzyme and the enzyme are prescribed in separate parts and mixed before measurement. The first reagent, the second reagent, and the coenzyme may be stored in separate vials, or can be used by dissolving in a solution (usually a buffer) at the time of use. The combination of the compositions prescribed for each part requires the first reagent tyramine oxidase, the second reagent tyrosine decarboxylase, and the coenzyme from the reaction principle, but other reagent components have stability when coexisting. It can be set as appropriate in consideration.
The origin of the vitamin B6 enzyme used in the present invention is not particularly limited, and any origin of microorganisms, plants, or animals may be used as long as it requires a coenzyme reversibly for expression of activity and maintenance of structure. A thing can also be used. Various commercially available products can be used. Alternatively, it can also be obtained by purifying a culture obtained by culturing cells (such as microorganisms) that produce the enzyme by a known method. Furthermore, it can also be produced using a cell-free protein synthesis system using wheat germ extract or the like. In addition, if the amino acid sequence of the enzyme obtained as described above is modified by a known method or chemically modified, it can be reversibly required for the expression of activity or maintenance of the structure. Can be used without any problems. Alternatively, an enzyme that does not require an electrolyte for its activity expression or maintenance of its structure in a natural state by modifying it by a known method and requires an electrolyte can also be used in the present invention.

Examples of the dye system that is visualized by reacting with hydrogen peroxide generated in the presence of peroxidase include an oxidizing coloring reagent, a reducing coloring reagent, a Trinder reagent, and a coupler thereof. As an oxidative coloring reagent,
Hexa (2-hydroxy-3-sulfopropyl) -4,4,4-triaminotriphenylmethane,
Hexa (3-sulfopropyl) -4,4,4-triaminotriphenylmethane,
Bis (2-hydroxy-3-sulfopropyl) toluidine and the like can be mentioned.

  Examples of the reducing coloring reagent include tetrazolium salts such as INT, MTT, TB, WST-1, WST-3, and WST-5, and are used in combination with an electron carrier such as 1-m-PMS. . As the Trinder reagent, N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3 , 5-dimethylaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) ) -M-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N -Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylmethylaniline, N-ethyl-N- (2-hydroxy-3 Sulfopropyl)-m-toluidine, include N- sulfopropyl aniline. Examples of the coupler include 4-aminoantipyrine and MBTH.

  Although it does not specifically limit as a buffering agent used for this invention, A tris buffer, a citrate buffer, a phosphate buffer, a borate borax buffer, a GOOD buffer, etc. are mentioned. Among these, Tris buffer, citrate buffer, borate buffer, and phosphate buffer have the advantage of being inexpensive, although the pH is likely to vary depending on the concentration and temperature. Examples of the GOOD buffer include MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS.

Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.
Example 1
The following tyrosine measurement reagents were stored at 10 ° C. for 7 days, and the remaining activity of tyrosine decarboxylase (apo-type enzyme) (ratio of activity value after storage to activity value immediately after preparation) was examined. In the comparative example, a reagent prepared by adding 40 μmol / L of pyridoxal 5-phosphate to the second reagent without adding the first reagent, pyridoxal 5-phosphate, was examined in the same manner.
(Preparation of reagents)
A tyrosine measuring reagent having the following composition was prepared.
First reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
TOOS 1g / L
Flavin adenine dinucleotide 2Na salt 8 μmol / L
Peroxidase (Toyobo PEO-301) 3 U / mL
Ascorbate oxidase (Toyobo ASO-311) 1 U / mL
Tyramine oxidase (Toyobo TYO-301) 0.3 U / mL
Pyridoxal 5-phosphate ester 10 μmol / L
Second reagent Mcylvine buffer 50 mM pH 6.0
Triton X-100 1g / L
4-Aminoantipyrine 0.3g / L
Tyrosine decarboxylase 1.2U / mL

Results: Shown in Table 1. In the comparative example, the remaining activity of tyrosine decarboxylase after 7 days at 10 ° C. decreased to 59%, whereas in the examples, good stability of 90% or more was exhibited after storage.
(Example 2)
The tyrosine measuring reagent of Example 1 was stored at 10 ° C. for 11 days, purified water and 200 μmol / L tyrosine aqueous solution were measured as samples by the following measurement method, and the purified water measured absorbance was subtracted from the 200 μmol / L tyrosine aqueous solution measured absorbance. The absorbance (sensitivity) was calculated and compared with the absorbance immediately after preparation to determine the relative percentage. In the comparative example, a reagent prepared by adding 40 μmol / L of pyridoxal 5-phosphate to the second reagent without adding the first reagent, pyridoxal 5-phosphate, was examined in the same manner.
(Measurement method)
A Hitachi 7170 automatic analyzer was used. First reaction was performed by adding 200 μL of the first reagent to 2.5 μL of the sample and incubating at 37 ° C. for 5 minutes. Thereafter, 50 μL of the second reagent was added and incubated for 5 minutes to form a second reaction. The absorbance at 570 nm was measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for the liquid volume and the difference between the absorbances was taken.
The result was calculated from the measured absorbance of purified water and 200 μmol / LL-tyrosine aqueous solution, and obtained as the tyrosine concentration.

Results are shown in Table 2. In the comparative example, the sensitivity after 11 days at 10 ° C. decreased to 52%, whereas in the example, after storage, it was almost 100%, indicating good stability.
(Example 3)
Immediately after the preparation of the tyrosine measurement reagent of Example 1 and each of the reagents stored at 10 ° C. for 7 days, the simultaneous reproducibility was examined by the measurement method described in Example 2. The simultaneous reproducibility was calculated by repeatedly measuring n = 20 and adding a coefficient of variation CV (%) to a sample prepared by adding L-tyrosine to commercial control serum QAP Troll IIX (Sysmex) to a final concentration of 100 μmol / L. . In the comparative example, a reagent prepared by adding 40 μmol / L of pyridoxal 5-phosphate to the second reagent without adding the first reagent, pyridoxal 5-phosphate, was examined in the same manner.

Results are shown in Table 3. In the comparative example, the coefficient of variation after 10 days at 10 ° C. deteriorated immediately after the preparation, whereas in the example, after storage, it was almost the same as that immediately after the preparation and showed good stability and precision.

  The method for stabilizing the vitamin B6 enzyme of the present invention can be used in fields of application such as reagents and in vitro diagnostics using the enzyme, and contributes greatly to the industry.

Claims (4)

A reagent having at least two parts including tyrosine decarboxylase and pyridoxal 5-phosphate , wherein tyrosine decarboxylase and pyridoxal 5-phosphate are formulated in separate parts. The reagent according to claim 1 , wherein the reagent using tyrosine decarboxylase is a tyrosine measuring reagent. A method of stabilizing a pyridoxal 5-phosphate in the reagent containing tyrosine decarboxylase and pyridoxal 5-phosphate, a configuration during storage, characterized by formulating the tyrosine decarboxylase and pyridoxal 5-phosphate separately, Method. The method according to claim 3 , wherein the reagent using tyrosine decarboxylase is a tyrosine measuring reagent.