JP3143050B2 - Stabilized glucose 6-phosphate dehydrogenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing glucose-6-phosphate dehydrogenase by chemical modification, and a method for stabilizing glucose-6-phosphate dehydrogenase in a clinical test method using glucose-6-phosphate dehydrogenase. The present invention relates to a highly stable measurement reagent for clinical tests comprising an acid dehydrogenase and a measurement method thereof.

[0002]

2. Description of the Related Art Glucose 6-phosphate dehydrogenase is an enzyme that catalyzes a reaction that produces glucose 6-phosphate and NADH or NADPH in the presence of a coenzyme NAD or NADP, or vice versa. It is widely distributed in the flora and fauna. Utilizing this property, glucose-6-phosphate dehydrogenase is used for quantification of glucose-related substances in an analysis sample and for enzyme activity of related enzymes, but has a problem in stability.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a technique for improving the stability of a glucose-6-phosphate dehydrogenase using glucose-6-phosphate as a product in a liquid clinical test reagent. With the goal.

[0004]

Means for Solving the Problems In view of the above conventional problems, the present inventors have conducted intensive studies on glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PD), and as a result, have found that leuconostoc, a kind of lactic acid bacteria, In G6PD of the genus,
A technique for improving the stability of the enzyme by chemically modifying it with a bivalent crosslinking reagent has been discovered. A liquid clinical test reagent using glucose-6-phosphate (hereinafter sometimes abbreviated as G6P) as a product and its measurement The system method was completed.

[0005] An example of a method for producing G6PD stabilized by glutaraldehyde modification of the present invention is shown below.

1) Purified G6PD derived from Leuconostoc mescentroides, 6 mg protein in 2.4 ml of 20 mM
K-PO ₄ buffer (pH 7.0, 0.2 M NaC
1, containing 0.5 mM EDTA). Then, while stirring vigorously with a mixer, the final concentration is 0.05
%, 12 μl of a 10% glutaraldehyde solution (manufactured by Wako Pure Chemical Industries, Ltd., for an electron microscope) was added dropwise. Then, it was left still at 25 ° C. for 30 minutes.

2) An equivalent amount of a 0.2 M glycine solution (pH 7.0) was added to the glutaraldehyde reaction solution, and the mixture was mixed to stop the cross-linking reaction. Then, it was left still for 30 minutes.

3) In order to remove low-molecular-weight substances from the reaction mixture finally showing a pale yellow color, a commercially available column (Biogel-10DG, manufactured by Bio-Rad) is used.
0 mM K-PO ₄ buffer (PH7.0,0.5MM
EDTA, containing 0.02% NaN ₃ ). Alternatively, dialysis may be performed in the above phosphate buffer instead of the desalting operation.

4) Further, in order to obtain only stabilized G6PD from the cross-linking reaction solution, in order to inactivate and remove unmodified enzymes and those which have been insufficiently cross-linked, The heat treatment operation may be performed in the step 2) or the step 3). Separation and purification means such as ion exchange chromatography may be employed after step 3).
Of course, the heat treatment and the chromatography operation can be combined. G6P with a bivalent crosslinking reagent
During the D cross-linking reaction, a substrate and a substrate analog or an inhibitor which cannot substantially react with the divalent cross-linking reagent used for protecting the enzyme active site may be added.

[0010] G6PD derived from Leuconostoc sp.
An enzyme preparation obtained by purifying from a cell culture or a recombinant enzyme obtained by transducing Escherichia coli or the like with the G6PD gene derived from Leuconostoc may be used.

The bacterial genus Leuconostoc according to the present invention may be any one such as Leuconostoc mententroides. As a representative strain of this microorganism, Leuconostoc mentencentroides is ATCC
12291.

The enzymatic activity of G6PD was calculated spectrophotometrically at 1 ° C./minute of NADH at 30 ° C.
The amount produced was defined as the enzyme activity (1 unit).

Measurement equipment: Hitachi spectrophotometer U, 2000 type Measurement temperature: 30 ° C. Measurement wavelength: 340 nm Substrate reaction solution composition: 0.1 M Tris-HCl buffer (pH 7.8) 3 mM MgCl ₂ 3 mM G6P 2 mM NAD ⁺

G by the above-mentioned glutaraldehyde modification
Table 1 shows the total activity value and the yield at each step in the stabilization operation of 6PD.

[0015]

[Table 1] 処理 Treatment process Total activity value (U) Yield (%) ─ ─────────────────────────────── Initial starting material 4,990 100 Glutaraldehyde modification reaction 1,167 23.4 50 ° C Heat treatment for 15 minutes 833 16.7 Fractionation with DEAE-5PW column 924 18.5────────────────────────────── ──

The method for measuring the thermal stability of G6PD stabilized by glutaraldehyde modification can be easily tested, for example, in the following manner.

Heat treatment temperature: 45, 50, 55 ° C. Heat treatment time: sampled at 0, 2, 5, 10, 20, and 30 minutes Added enzyme concentration: 0.2 U / ml Buffer: 50 mM K-PO ₄ (pH 7.0) 0) 0.5 mM EDTA 0.1% BSA 0.02% NaN ₃ Calculation method of thermal stability: The ratio of the residual activity after the heat treatment to the initial G6PD activity value is plotted against the heat treatment time to stabilize. The half life of G6 PDH is determined.

The enzymatic properties of G6PD stabilized by glutaraldehyde modification were compared with the untreated enzyme based on the measurement of the molecular weight of the cross-linked enzyme by gel filtration and the degree of reaction with G6P as a substrate. For the composition for measuring the Km value for G6P, see G6PD described above.
One according to the enzyme activity reagent composition and clinical chemistry (199
0) The measurement was performed in accordance with the final reaction solution composition for measuring CK (creatine kinase) activity described in Vol. 19, No. 2, 185-208. Table 2 shows the obtained comparative analysis results.

[0019]

[Table 2] Enzymatic properties Unmodified G6PD Modified G6PD Ｋ Molecular weight (gel filtration method) Km value for 130KD 130KD G6P (30 ° C, pH 7.8, 0.1 mM 0.15 mM 2 mM NAD ⁺ during ^{measurement) 1) (37 ℃, pH6.7} , 0.1mM about 0.2 mM CK activity measurements in the reagent composition) ²⁾ ────────────────── ─────────────

In the present invention, various divalent crosslinking reagents used for stabilizing G6PD include various substances. Table 3 shows the results of the thermostability test of the modified enzyme together with an example. In addition to the crosslinking reagents listed in the following table, disuccinimidyl glutarate (DSG), disuccinimidyl tartrate (DST) and the like can be mentioned.

The modification reaction conditions G6PD by crosslinking reagent; G6PD: Leuconostoc from 1.4mg protein / ml (about 1000 U / ml) crosslinked reagent concentration: 5 mM reaction solution composition: 20mM K-PO ₄ buffer ( (pH 7.0) 0.2 mM NaCl 0.5 mM EDTA 0.02% NaN ₃ Reaction conditions: 25 ° C., 20 minutes Reaction terminator: Add 0.1 M glycine (pH 7.0), mix and then stand for 15 minutes

[0022]

Table 3 Bivalent cross-linking used for G6PD modification Thermostability of modified enzyme after reaction Cross-linking reagent (5 mM in reaction solution) Enzyme activity recovery rate (50 ° C, 20 minutes) ──────────────────── ──────────────── Unmodified 100% 38.5% Glutaraldehyde 9.7 71.5 Dimethyladipic acid 87.5 40.6 Dimethylsuberimidate 81.2 39. 2 disuccinimidyl suberate 33.9 50.5 dithiobissubinimidylpro << 1.0-pionate dimethyldithiobispropion 58.3 42.2 imidate ethylene glycobissuccinimi << 1.0- − Jill succinate ───────────────────────── ───────────

Now, the present invention will be described in further detail with reference to Examples. The present invention is not limited to the embodiments.

[0024]

Example 1 1) Purified G6 derived from Leuconostoc mesenteroides
PD, 6 mg protein in 2.4 ml of 20 mM K-PO ₄
Buffer (pH 7.0, 0.2 mM NaCl, 0.5 m
M EDTA). Then, while vigorously stirring with a mixer, 12 μl of a 10% glutaraldehyde solution (manufactured by Wako Pure Chemical Industries, Ltd., for an electron microscope) was added dropwise to a final concentration of 0.05%. Then, at 25 ° C, 3
Let stand for 0 minutes.

2) An equal amount of a 0.2 M glycine solution (pH 7.0) was added to and mixed with the above glutaraldehyde reaction solution to stop the cross-linking reaction. Then, it was left still for 30 minutes.

3) In order to remove a low-molecular-weight substance from the reaction mixture finally showing a pale yellow color, a commercially available column (Biogel-10DG, manufactured by Bio-Rad) was used.
0 mM K-PO ₄ buffer (PH7.0,0.5MM
EDTA, containing 0.02% NaN ₃ ).

4) Since the glutaraldehyde cross-linking reaction mixture in the above step 2) contains an unmodified enzyme in which a cross-linking reaction has not been formed and an enzyme in an inadequate state, etc. At a temperature of 55 ° C. with respect to the reaction mixture of the above 2) for the purpose of obtaining only stabilized G6PD.
Heat treatment was performed for 15 minutes.

In the following examples, those up to the above step 3) are referred to as modifying enzyme (# 1), and those subjected to heat treatment after the crosslinking reaction are referred to as modifying enzyme (# 2, heat treatment). I decided to do it.

[0029]

Example 2 The method for measuring the thermal stability of G6PD stabilized by glutaraldehyde modification was tested as follows.

Heat treatment temperature: 45, 50, 55 ° C. Heat treatment time: 15 minutes Concentration of added enzyme: 0.2 U / ml Buffer for enzyme heat treatment: 20 mM K-PO ₄ (pH 7.0) 0.5 mM EDTA 0.2 M NaCl Calculation method of 0.1% BSA 0.02% NaN ₃ thermal stability degree: The ratio of the residual activity after the heat treatment to the initial G6PD activity value is shown for various treatment temperatures.

[0031]

EXAMPLE 3 CP of Modified G6PD Stabilized in Example 1
The storage stability in the reagent composition for K activity measurement was examined in the following manner. The results are shown in FIG.

Storage temperature: 4 ° C. and 37 ° C. Storage days: Sampling on days 0, 3, 6, and 9 G6PD concentration added: 0.2 U / ml Lysis buffer: 100 mM Imidazole acetate buffer (pH 6.7) 2 mM EDTA 10 mM magnesium acetate Reagent composition: 2.5 mM ADP 25 mM NAC 6.25 mM AMP 12.5 μM AP ₅ A 2.5 mM NADP ⁺ no glucose added or 20 mM glucose added Calculation method of thermal stability degree: storage for initial G6PD activity value The activity ratio is plotted against the number of storage days, and the half-life of the stabilized G6PDH is determined based on the residual activity ratio.

[0033]

Example 4 CP of Modified G6PD Stabilized in Example 1
Following enzyme performance at the time of K activity measurement was investigated in the following manner. The reagent composition assembled to measure CPK activity is described in Clinical Chemistry (1990) Vol. 1, No. 2, 185-208.
The measurement was performed in accordance with the final reaction solution composition for measuring CK (creatine kinase) activity described on the page. The results are shown in FIG.

Specimen: Commercially available control serum (Moni-Toll II manufactured by Baxter) Specimen blank: 0.9% NaCl aqueous solution Measuring instrument: Corbus Fara Measuring temperature: 37 ° C. Addition of specimen and measuring reagent: Specimen: pure water: first reagent : 2nd reagent = ratio 6: 4: 240: 60 (μl) Calculation of CPK activity value: After adding and mixing a sample and a measurement reagent to start a reaction, in 1 minute from 2.5 to 3.5 minutes ΔA340 / min was determined from the rate assay. CPK activity measurement value and G6: Relationship between apparent ΔPDH following ability obtained from time course from the start of the reaction A340 / min was plotted as a relationship with the amount of G6 PDH enzyme added to the first reagent composition. CPK activity measurement reagent dissolution: 100 mM imidazole acetate buffer (pH 6.7) Buffer 2 mM EDTA 10 mM magnesium acetate 20 mM glucose First reagent: 2.5 mM ADP 25 mM N-acetylcysteine 6.25 mM AMP 12.5 μM AP ₅ A 5 mM NADP ⁺ 3 U / mL HK Second reagent: 150 mM creatine phosphate

[0035]

Example 5 A calibration curve was prepared for measuring CPK activity using a modified G6PD.

Specimen: Rabbit skeletal muscle-derived creatine kinase dissolved in 2,000 U / L and diluted in 5 steps and used. CK lysis buffer: 1% BSA 10 mM NAC 0.05% NaN ₃ 50 mM Tris HCl buffer ( pH 8.5)

In preparing a CPK calibration curve using creatine kinase derived from rabbit skeletal muscle, 2 U / ml of stabilized G6PD was added to the first reagent composition. Other conditions for measuring the CPK activity were the same as in Example 4. The results are shown in FIG.

[0038]

Industrial Applicability The present invention relates to a liquid clinical test reagent used in a method for measuring a biological substance using glucose 6-phosphate as a product, and G6P chemically modified with a divalent crosslinking reagent.
By using D, the stability of the enzyme can be significantly improved.

[Brief description of the drawings]

FIG. 1 is a chart showing the thermal stabilization of Leuconostoc G6PD by a glutaraldehyde crosslinking reagent.

FIG. 2 (a) is when glucose is not added, (b) is 2
5 is a table showing the storage stability of a G6PD stabilized by a cross-linking treatment in a reagent composition for measuring CPK activity when 0 mM glucose is added.

FIG. 3: C of G6PD stabilized by cross-linking treatment
5 is a table showing the following enzyme performance at the time of PK activity measurement.

FIG. 4 is a chart showing a calibration curve of a reagent for measuring CPK activity using G6PD stabilized by a crosslinking treatment.

Claims (3)

(57) [Claims] 1. A measuring reagent using glucose-6-phosphate dehydrogenase, wherein the glucose-6-phosphate dehydrogenase comprises:
A measuring reagent characterized by being glucose-6-phosphate dehydrogenase stabilized by being chemically modified with a bivalent crosslinking reagent. 2. A measuring reagent using glucose-6-phosphate dehydrogenase, wherein said glucose-6-phosphate dehydrogenase comprises:
A measuring reagent characterized by being glucose-6-phosphate dehydrogenase stabilized by heat treatment after being chemically modified with a bivalent crosslinking reagent. 3. A method for measuring a biological component with a measuring reagent using the stabilized glucose-6-phosphate dehydrogenase according to any one of claims 1 and 2.