JP2646911B2 - Method for stabilizing xanthine oxidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for stabilizing xanthine oxidase (hereinafter abbreviated as XOD) in a solution.

[0002]

2. Description of the Related Art XOD is present in buttermilk, animal organs, bacteria, etc., and is known as an enzyme that oxidizes purine bases such as xanthine and hypoxanthine to generate uric acid. It is widely used in the field of clinical biochemical testing by the method. For example, guanase (hereinafter abbreviated as GUA), adenosine deaminase (hereinafter abbreviated as ADA), superoxide dismutase (hereinafter abbreviated as SOD), and inorganic phosphorus (hereinafter abbreviated as IP). XOD is becoming indispensable in measurement.

[0003] However, since XOD is an enzyme that is extremely unstable (easy to deactivate) in a normal buffer, XOD is
Measuring solution using, for example, GUA, ADA, SO
The reagents for measurement such as D and IP have a problem that storage stability after preparation is poor.

To solve this problem, as a method for stabilizing XOD, a method in which phosphoric acid, protein, acidic amino acid, aliphatic carboxylic acid and the like coexist (Japanese Patent Laid-Open No. Sho 62-550).
No. 81) has been proposed, but it is not an effective method for stabilizing XOD in a solution, although the effect of stabilizing a freeze-dried product is recognized. Further, a method of coexisting an aromatic carboxylic acid and / or a boron compound in an XOD solution (Japanese Patent Application Laid-Open No. 62-210988) has also been proposed, but this method stabilizes a solution itself containing XOD,
It is effective for stabilization of a test solution containing XOD in a system for measuring SOD activity, but in a system for measuring GUA activity, ADA activity, IP, etc., there is a restriction on the concentration of stabilizer used. In addition, there is a problem that a large excess of XOD is required, and the development of a more effective stabilization method is urgently desired.

[0005]

An object of the present invention is to provide a method for effectively stabilizing XOD in a solution, which has been made in view of the above-mentioned circumstances, and is also applicable to measurement systems such as GUA, ADA, SOD, and IP. An object of the present invention is to provide a method for stabilizing XOD which can be used effectively.

[0006]

The present invention relates to coexistence of a salt (including a complex salt) of a metal selected from the group consisting of iron, copper, cobalt and manganese or / and a chelate compound of these metals in a solution containing XOD. An invention of a method for stabilizing an XOD characterized by the following.

Namely, the present inventors found that the course of extensive studies on how to stabilize the XOD in solution, generally in many heavy metal ions of inhibiting the enzyme activity, iron, copper, co
The present inventors have found that salts of baltic and manganese (including complex salts) or chelating compounds of these metals have an unexpectedly stabilizing effect on XOD in a solution, thereby completing the present invention.

[0008] Salts (including complex salts) of metals selected from the group consisting of iron, copper, cobalt and manganese used in the present invention and chelate compounds of these metals (hereinafter abbreviated as salts according to the present invention). ) Is the XOD in the solution
There is no particular limitation as long as it has the effect of stabilizing the compound. Specifically, for example, ferrous chloride, ferric chloride,
Salts such as ammonium ferrous sulfate, ammonium ferric sulfate, manganese chloride, cupric chloride, and cobalt chloride, such as potassium ferricyanide, kalim ferrocyanide, iron (III) -acetylacetonate, Mn-acetylacetonate, Complex salts such as Co-acetylacetonate,
Chelating compounds obtained from iron, copper, cobalt, and manganese and a chelating agent such as ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), diaminopropanetetraacetic acid, hydroxyethylethylenediaminetriacetic acid, and glycoletherdiaminetetraacetic acid are exemplified. Among them, potassium ferrocyanide, EDTA-Fe (II
I) and the like are preferred. Further, as is clear from the above examples, the ionic value of iron or the like does not cause any particular problem in the present invention.

The concentration of the salt or the like according to the present invention varies depending on the kind and combination of the salt and the chelate compound used, but is usually 0.01 ppm or more, preferably 0.1 ppm or more in the solution containing XOD. It is. The upper limit of the concentration of the salt or the like according to the present invention also varies depending on the type and combination of the salt or chelate compound used, but in setting the concentration of the use, the stability of other enzymes coexisting in the solution containing XOD is reduced. Should be considered, and the effect on the color reaction such as coloring of the reagent and blind increase. In addition, when a cyanide or the like is used, its use concentration should be determined based on the emission standard, and when a sufficient effect can be expected at a low concentration, it is not necessary to use an unnecessarily large amount. Not to mention nothing. Considering the above conditions, the optimal working concentration of these compounds is usually 0.01 to 500.
It is appropriately selected from the range of ppm, preferably from the range of 0.1 to 200 ppm.

The salts and the like according to the present invention may be used alone or in an appropriate combination of two or more kinds. When used in combination, there are no particular restrictions on the compounding ratio of each compound. Absent.

The origin of the XOD which can be stabilized by the method of the present invention is not particularly limited, and for example, those derived from buttermilk, animal organs, bacteria, etc. are usually used in the field of clinical biochemical examination. Are preferably mentioned. Further, the stabilizing effect of the method of the present invention does not change depending on the XOD concentration in the solution, but a measuring solution containing XOD known per se usually used in the field of clinical biochemical examination, for example, GUA. , ADA, SOD, IP
, Or even higher or lower,
Both are effective.

The pH of the solution containing XOD which can be stabilized by the method of the present invention is not particularly limited as long as it does not deactivate the XOD itself.
It is selected from the range of 9.0. In addition, the solution contains a measuring solution containing XOD known per se, for example, GUA, AD
A, SOD, buffer solution such as Good's buffer, phosphate, tris (hydroxymethyl) aminomethane, etc., surfactants, saccharides, proteins, preservatives contained in the test solution for SOD, IP, etc. Needless to say, an agent may be included. In addition, the concentrations of these components other than XOD in the solution may be appropriately determined according to the concentrations in the measurement test solution containing XOD known per se.

In order to carry out the method of the present invention, for example, one or more of the salts and the like according to the present invention may be appropriately selected and coexisted in a solution containing XOD at the above-mentioned concentration. Well, no special operation is required. As a method for allowing the salts and the like according to the present invention to coexist in a solution containing XOD, for example, a method of adding them in advance to a freeze-dried preparation prepared as a measuring reagent such as GUA, ADA, SOD, IP, or the like A method of adding the reagents for measurement as described above to a solution for dissolving a lyophilized preparation, etc., as long as the salts and the like according to the present invention can be finally coexisted in a solution containing XOD. Either may be used.

The solution containing XOD stabilized by the method of the present invention can be stored for a longer period of time than conventional ones. In addition, the method of the present invention employs GUA, AD using XOD.
It can also be used very effectively as a method for stabilizing a test solution for measurement such as A, SOD, and IP.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0016]

【Example】

Embodiment 1 FIG. XOD (from buttermilk) 0.35U / m
l, 100 mM N- (2-acetamido) -2-aminoethanesulfonic acid buffer (pH: 0.8% containing bovine serum albumin 0.8% and 25 ppm of a salt or the like according to the present invention)
6.5) was prepared and used as an XOD solution.

The obtained XOD solution was stored at 5 ° C. for a predetermined number of days, and changes in XOD activity were monitored. Table 1 shows the results.
The numerical values in the table are the relative values when the enzymatic value immediately after preparation is defined as 100 for the residual activity value of the measured value. The measurement of the XOD activity was carried out in a 0.1 M tris (hydroxymethyl) aminomethane buffer (pH 7.5).
Hypoxanthine 0.1 mM, disodium ethylenediaminetetraacetate 0.1 mM, 4-aminoantipyrine 0.0
A solution containing 5 mM, 1 mM of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium salt and 5 u / ml of peroxidase was prepared and used as a substrate-color developing reagent. -Take 2.2 ml of the coloring reagent solution, heat it in a 37 ° C constant temperature bath for 12 minutes, add 10 µl of the sample, measure the change in absorbance at 37 ° C and a wavelength of 600 nm (2 minutes), and measure the linear portion per minute. Was calculated based on this value.

Comparative Example 1 XOD (from buttermilk)
0.35 U / ml, bovine serum albumin 0.8% and Na
₂ MoO ₄ , CaCl ₂ , MgCl ₂ , EDTA-2N
a, EDTA-Ni (II) or EDTA-Zn (I
100 mM N- (2-acetamido) -2-aminoethanesulfonic acid buffer solution (pH 6.
5) were prepared, and X
Changes in OD activity were tracked. The results are shown in Table 1.

[0019]

As is evident from the results in Table 1, when the salts and the like according to the present invention coexist in the XOD solution, the XOD
It can be seen that the stability during storage is improved.

Experimental Example 1. 100 mM N- (2-acetamido) -2-aminoethanesulfonic acid buffer solution (pH
6.5) XOD (from buttermilk) 0.35U / m
l, bovine serum albumin 0.8% and K ₄ [Fe (CN)
₆ ] [or a predetermined concentration of EDTA-Fe (III)] was dissolved to prepare an XOD solution, and the obtained XOD solution was followed for changes in XOD activity in the same manner as in Example 1. Table 2 shows the results. The numerical values in the table are relative values when the residual activity value of the measured value is defined as 100, which is the enzyme activity value immediately after preparation. The measurement of the XOD activity was performed in the same manner as in Example 1.

[0022]

As is clear from the results in Table 2, X in the solution
It can be seen that in order to stabilize the OD, it is necessary that the salts and the like according to the present invention coexist in the solution in an amount of 0.01 ppm or more, preferably 0.1 ppm or more.

[0024]

As described above, according to the present invention, XOD in a solution which was difficult to be sufficiently stabilized by the conventional method
And a method that can effectively stabilize
This invention has a remarkable effect in that it can be used without any hindrance in stabilizing a reagent solution for measurement such as UA, ADA, SOD, and IP, and is a great invention that contributes to the industry.

Claims (1)

(57) [Claims] 1. A salt (including a complex salt) of a metal selected from the group consisting of iron, copper, cobalt and manganese and / or a chelate compound of these metals coexist in a solution containing xanthine oxidase. Xanthine oxidase stabilization method.