JP2551923B2 - Acetate kinase composition - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to acetate kinase compositions.

[0002]

2. Description of the Prior Art Acetate kinase (hereinafter abbreviated as AK) is

Embedded image It is an enzyme that catalyzes the use of adenosine triphosphate (hereinafter abbreviated as ATP), and is used as an enzyme of a measuring reagent for diagnosis and the like. For example, the following reactions as reagents for measuring acetic acid in test samples and diagnostic reagents The acetic acid derived by
It is applied to a reagent for measuring cholinesterase by acting TP and measuring the generated adenosine diphosphate by a known method, and its importance is increasing more and more. B. stearothermophilus and E.co as the origin of AK
li etc. are known. In particular, AK separated and purified from thermophiles such as B. stearothermophilus is also a thermostable enzyme, its properties are very good, and its thermal stability is guaranteed up to 65 ° C. However, it was found that many AKs lack stability when formed into a composition containing AK as a diagnostic reagent, and the stability is further impaired particularly when freeze-dried, etc. Had been an obstacle.

[0003]

SUMMARY OF THE INVENTION In view of the above, the present inventors have earnestly studied to find a stable AK composition, and as a result,
As a stabilizer, a compound having an SH group, in particular, a low molecular weight thiol compound was found, and the present invention was accomplished.

[0004]

That is, the present invention provides a measuring composition for measuring acetic acid in a test sample or acetic acid derived from a substance in a test sample, which comprises at least acetate acetate as a main component. The present invention relates to an acetate kinase composition, which comprises a low molecular weight thiol compound as a stabilizer.

Examples of the SH group-containing compound that can be used in the present invention include N-acetyl-cysteine, cysteine,
Cysteine-containing di- or tri-, such as glutathione
Peptides, low-molecular thiol compounds having a molecular weight of 310 or less, such as mono- or di-SH-substituted threitol or erythritol such as dithiothreitol or dithioerythritol, or lower alkyl thiols such as mercaptoethanol, and one or more of them. Can be used. From the viewpoint of stability, N-acetyl cysteine is preferable. As the buffering agent, a usual one can be used. When the pH of the aqueous composition exceeds 10, the stability of the thiol compound is deteriorated, and when it is less than 6, the enzyme in the system is harmed, so that it is usually 6 to 10. preferable. The concentration of the low-molecular-weight thiol compound used is 1 mM or more from the viewpoint of the stabilizing effect, but it is preferably 10 mM or more. Other components can be added to the AK-containing preparation of the present invention as necessary. The strong oxidant that oxidizes the low molecular weight thiol compound is excluded.

The AK-containing composition of the present invention can be used as an aqueous composition or as a freeze-dried composition without any problem in any form. Aqueous compositions containing AK allow the present invention to be used for extended periods of time, recognizing the advantages of the present invention. Further, the freeze-dried composition can be obtained by freeze-drying the AK-containing aqueous composition by an ordinary method, but the present invention does not cause the problem of enzyme inactivation in the freeze-drying step, and The stability of AK as the obtained freeze-dried composition is remarkably high. The stability of AK is remarkably enhanced even when it is an aqueous composition.

Next, the enzyme activity of AK was measured by the following method. Imidazole-hydrochloric acid buffer solution (pH 7.2) 71.25 mM ATP 12.5 mM PEP 4.25 mM magnesium chloride 25 mM NADH 0.325 mM potassium chloride 93.75 mM PK 12.5 units / ml LDH 13.75 units / ml (however, PEP Is phosphoenolpyruvate, NAD
H is reduced nicotinamide adenine dinucleotide, P
K is an abbreviation for pyruvate kinase, and LDH is an abbreviation for lactate dehydrogenase. ) 20 μl of AK enzyme solution was added to 2.0 ml of the reaction solution having the above concentration, preheated at 30 ° C. for 5 minutes, 0.5 ml of 2M sodium acetate solution was added to start the reaction, and the absorbance at 340 nm was measured at 1 minute. For 1 minute. ΔE: Absorbance change for 1 minute

[0008]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 Tris (human oxymethyl) aminomethane-maleic acid (pH 9.0) 100 mM magnesium sulfate 12.5 mM potassium chloride 31.25 mM ATP 6.25 mM PEP 1.25 mM NADH 0.325 mM AK 37.5 u / ml PK 12. 5 u / ml LDH 6.25 u / ml Each additive of a low molecular weight thiol compound was added to the above solution at the concentration shown in Table 1, and the results of storing at 25 ° C. for 4 days are shown in Table 1. However, the AK activity at the start of storage was set to 100%. As is clear from Table 1, the stabilizing effect was recognized in the solution composition.

[0009]

[Table 1] Storage stability in aqueous solution (25 ° C, 4 days) Additive concentration Residual activity (%) No addition-76 N-Acetylcysteine 2.5 mM 100 Glutathione 2.5 mM 89 Cysteine 2.5 mM 97

Example 2 AK 2 × 10 ⁴ units PK 1 × 10 ⁴ units LDH 1 × 10 ³ units ATP 1 g PEP 0.05 g NADH 0.14 g Tris-hydrochloric acid buffer solution (pH 8.0) 25 m
dissolve in 1. Furthermore, a freeze-dried stock solution containing a low-molecular thiol compound and other additives was prepared and freeze-dried. The stability of AK activity before and after freeze-drying was examined and is shown in Table 2. However, the enzyme activity of the stock solution before lyophilization is 100%
And As is clear from Table 2, AK was greatly stabilized by the low molecular weight thiol compound as compared with the case where no addition was made.

[0011]

[Table 2] Additive concentration Concentration of residual activity after lyophilization (%) No addition-66 N-acetyl cysteine 10 mM 95 glutathione 10 mM 93 cysteine 10 mM 92 dithiothreitol 10 mM 93 dithioerythritol 10 mM 90 glycerol 1% 58 bovine serum albumin 1% 69 Fructose diphosphate 10 mM 69 Coenzyme A 0.25 mM 68

Example 3 The results of storing the freeze-dried product of the present invention prepared in Example 2 at 37 ° C. for 1 month are shown in Table 3. However, the AK activity immediately after freeze-drying was set to 100%. As is clear from Table 3, when the thiol low-molecular weight compound was added, the degree of deactivation was very small, although it was largely deactivated without addition.

[0013]

[Table 3] Storage stability in FD product (37 ° C, 1 month) Additive concentration Residual activity (%) No addition-39 N-acetylcysteine 10 mM 96 Glutathione 10 mM 95 Cysteine 10 mM 93 Dithiothreitol 10 mM 94 Dithioerythritol 10 mM 93

Example 4 A lyophilized stock solution was prepared by changing the concentration of the low-molecular-weight thiol compound (N-acetylcysteine) having the stock solution composition of Example 2, and the AK activity before and after freeze-drying was measured. The result is shown in Figure 1.
Displayed on. As is clear from the graph of FIG. 1, when 1 mM of N-acetylcysteine was added, about 85% of residual activity was observed, and when it was adjusted to 10 mM or more,
Remaining activity of 0% or more is observed.

[0015]

As is apparent from the above, according to the present invention, there is provided an acetate kinase composition containing in a stable state an acetate kinase, which has conventionally been problematic in stability as a composition for various uses. Became possible.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the degree of residual AK activity and the concentration of a low-molecular-weight thiol compound (N-acetylcysteine) (in a freeze-dried stock solution) after freeze-drying of an AK-containing composition according to the present invention. Is.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuhiko Nagata 23 Uji Kozakura, Uji-shi, Kyoto Unitika Central Research Laboratories (72) Inventor Takaaki Matsuo 23 Uji Kozakura, Uji-shi Kyoto Prefecture Unitika Central Research Laboratories

Claims (1)

(57) [Claims] 1. A low-molecular-weight thiol compound as a stabilizer in a measuring composition for measuring acetic acid in a test sample or acetic acid derived from a substance in a test sample, which comprises at least acetate acetate as a main component. An acetate kinase composition comprising: