JP2868549B2 - Method for measuring diaphorase activity - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for measuring diaphorase activity using nitroblue tetrazolium (hereinafter abbreviated as NBT).

(Prior Art) Diaphorase is nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) or nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH) (hereinafter, when either one is meant, NAD
(P) H. ) Is an enzyme that catalyzes the reduction reaction of the dye.

As a method for measuring the activity of diaphorase, a colorimetric method in which a change in absorbance associated with reduction of a dye is measured using a spectrophotometer is generally used. As a dye reduced by diaphorase, dichlorophenol indophenol (hereinafter abbreviated as DCIP) and NBT are used.

In the activity measurement method using DCIP, the amount of decrease in absorbance at 600 nm is determined by reducing DCIP having an absorption at a wavelength of 600 nm with NAD (P) H and converting it into reduced DCIP having no absorption at a wavelength of 600 nm. Diaphorase activity (Biochem. J., 191: 457-4).
65, 1980). Therefore, it is necessary to increase the absorbance of the measurement solution itself at the beginning of the activity measurement. for that reason,
There was a drawback that the absorbance at 600 nm at the start of the activity measurement was not constant for each measurement and was slightly different. Furthermore, even when the enzyme is not present in the measurement solution, NAD (P) H non-enzymatically reduces DCIP, so that the absorbance at 600 nm decreases.

On the other hand, a method for measuring the activity of diaphorase using NBT, which measures the increase in absorbance at 550 nm of diformazan produced by reduction of NBT. Therefore, the absorbance at the start of measurement is zero in all the measurement solutions, and in this regard, the activity of diaphorase can be detected more accurately than using DCIP as a substrate. For these reasons, when diaphorase is used in applications where sensitivity and accuracy are important, such as enzyme immunoassay, NBT is used as a substrate rather than DCIP at present.

(Problems to be Solved by the Invention) The use of NBT as a substrate has the above advantages, but NBT is non-enzymatically converted to diformazan in the presence of NAD (P) H, and the absorbance at 550 nm is reduced. It is known to rise gradually. This non-enzymatic increase in absorbance is called a blank. Therefore, when measuring the activity of diaphorase using NBT, the increase in absorbance is measured for both the measurement solution containing diaphorase and the measurement solution containing no diaphorase, and the increase in the absorbance is obtained from the value obtained in the former. Is subtracted from the value. In other words, the former is measured as the sum of the increase in absorbance due to the diaphorase enzymatic reaction and the increase in absorbance due to the non-enzymatic action of the blank. Therefore, subtracting the blank value measured in the latter gives the true diaphorase activity. Can be calculated. In such an activity measurement method,
When the activity of a small amount of diaphorase is measured, the ratio of the blank to the color development of diformazan by the enzymatic reaction is increased, which makes it difficult to accurately measure the activity of diaphorase.

An object of the present invention is to provide an activity measurement method capable of obtaining an accurate diaphorase activity value even when the amount of diaphorase is small by suppressing a blank value.

(Means for Solving the Problems) As a result of intensive studies to solve the above problems, the present inventors have found that ethylenediaminetetraacetic acid (hereinafter referred to as E) is contained in the measurement solution.
Abbreviated as DTA. ) Was found to reduce the blank value, and arrived at the present invention.

That is, the gist of the present invention is a method for measuring the activity of diaphorase, characterized in that EDTA is contained in a measurement solution when measuring the activity of diaphorase using NBT.

 Hereinafter, the present invention will be described in detail.

In the activity measurement method of the present invention, a sample containing diaphorase and components such as NBT, EDTA, NAD (P) H, a surfactant and a buffer are mixed to form a measurement solution, and diformazan formed by reduction of NBT is prepared. What is necessary is just to measure the amount of increase in the absorbance.

As the EDTA used in the present invention, any one such as salt-free EDTA, potassium salt, sodium salt and the like can be used. At this time, the EDTA concentration in the measurement solution was 1 to 50 mM.
And more preferably 3 to 10 mM.

As the components other than EDTA used in the present invention, those used in the conventional activity measurement method are used as described below as conventional.

The buffer used in the present invention includes phosphate buffer,
A buffer having a buffer action near neutrality such as a triethanolamine buffer can be used.
10 is preferable, and 6 to 8 is more preferable. At this time, the concentration of the buffer solution in the measurement solution was 10 to 500 mM.
Is more preferable, and more preferably 50 to 100 mM.

As the concentration of the NBT used in the present invention in the measurement solution,
It is preferably 0.1 to 10 mM, more preferably 0.2 to 1 mM.

The NAD (P) H used in the present invention includes NADH or NADH.
Any of DPH may be used, but NADH is preferred. The concentration of NAD (P) H in the measurement solution at this time is preferably 0.1 to 5 mM, more preferably 0.5 to 2 mM.

The type of the surfactant used in the present invention is not particularly limited, but a triton-based surfactant is preferable, and Triton X-100 is more preferable. At this time, the concentration of the surfactant in the measurement solution was 0.05 to 5 W / V.
%, More preferably in the range of 0.1 to 0.5 W / V%.

In the activity measurement method of the present invention, diaphorase and the above-described components need only be present in the measurement solution at the start of measuring the amount of increase in absorbance, and the order of mixing them is not particularly limited.

The activity measurement method of the present invention can be specifically carried out, for example, as follows.

That is, the sample containing diaphorase is preferably prepared so that the concentration of diaphorase in the measurement solution is 0.002 to 0.2 units / ml. For this purpose, for example, if necessary, the sample may be diluted with a buffer having a buffering action near neutrality so that the concentration of diaphorase in the sample becomes 2 units / ml or less, and the sample may be used at 100 μm or less.

A buffer containing the above concentrations of EDTA, NAD (P) H, and surfactant was added to a sample containing such a diaphorase for 80 minutes.
Add about 0-900μ to make the total amount 900μ.

The solution is then pre-warmed to 20-40 ° C. The preliminary heating time at this time may be a time for the solution to reach the set temperature, and is usually preferably about 3 to 5 minutes.

Then, 1 to 100 mM, preferably 2 to 10 mM NBT solution,
Add 100 μl to the above solution as volume.

 After this, colorimetry may be performed.

The colorimetric method used in the present invention includes a rate assay method in which the absorbance is measured over time, and a reaction performed after the enzyme reaction has been carried out for a certain period of time, the reaction is stopped, the absorbance is measured, and the enzyme activity is measured based on the change in the absorbance. An end point assay method for calculation is used, and in each case, the absorbance at a wavelength of 520 to 550 nm can be measured using a commercially available spectrophotometer. To stop the reaction in the end point method, for example, hydrochloric acid is used, and the pH of the measurement solution is preferably adjusted to 3 or less.

The blank measurement may be performed in the same manner as described above except that diaphorase is omitted.

The diaphorase that can be measured by the activity measuring method of the present invention is not particularly limited, and examples thereof include those derived from bacteria and those derived from animal tissues.

One unit of diaphorase activity is defined as the amount of enzyme that reduces 1 μmol of NBT per minute at pH 7.0 and 30 ° C.

(Examples) Next, the present invention will be described specifically with reference to examples.

Reference Example 1 First, a solution having the following composition was prepared.

500 mM triethanolamine-HCl (pH 7.0) 60 µ 20 mM NADH 60 µ 1% Triton X-100 60 µ H ₂ O 300 µ Next, add any of 0, ₂₀ , 50 and 100 mM EDTANa ₂ to 60 µ (Final concentrations of 0,2,5,
It becomes 10 mM. ). After incubating this at 30 ° C for 3 minutes, 60 mM of 5 mM NBT was added and reacted at 30 ° C. The increase in absorbance at 550 nm per minute was measured, and the effect of adding EDTA to suppress the blank was examined.

 The results are shown in Table 1 and FIG.

As shown in Table 1 and FIG. 1, the blank decreased as the EDTA concentration increased, and the blank decreased by about 2.5 times when EDTA was not added and the final concentration of EDTA was 5 mM.

Example 1 As shown in Reference Example 1, the following two types of diaphorase activities were measured, with and without EDTA added, so that the final concentration at which blank suppression effect was observed was 5 mM. Solutions were prepared, and the amount of diaphorase added was changed for each.

500 mM triethanolamine-HCl (pH 7.0) 60 µ 20 mM NADH 60 µ 1% Triton X-100 60 µ 0 or 50 mM EDTANa ₂ 60 µ H ₂ O 290 µ 0,1.0,2.0,3.0,4.0 ng diaphorase (Bacillus stearothermophilus) Origin, 120 units / mg), a solution containing either 10μ Next, after incubating these at 30 ° C for 3 minutes, 5mM
After adding 60μ of NBT and reacting at 30 ° C for 10 minutes, 1N HCl
The reaction was stopped by adding 150 μ, and the absorbance at 550 nm was measured. The results are shown in Table 2 and FIG.

As is clear from Table 2 and FIG. 2, the blank (in the table, the value of 0 ng of diaphorase) in EDTA-added (inventive) was reduced to 5/9 compared with EDTA-free (in the prior art). The enzyme activity (the value obtained by subtracting the blank from each value) hardly changed, and the ratio of the blank to the total absorbance was reduced, so that more accurate diaphorase detection was possible.

(Effect of the Invention) The activity measurement method of the present invention suppresses blanks,
Accurate diaphorase activity can be measured.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between EDTA concentration and blank.
FIG. 2 is a diagram showing the relationship between the amount of diaphorase and the absorbance. In FIG. 2, the open circles indicate diaphorase activity values (in the present invention) including blanks when EDTA was added, and the open circles indicate E
The diaphorase activity value including the blank when DTA was not added (prior art) is shown.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-1-181798 (JP, A) JP-A-63-52897 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12Q 1/00-3/00

Claims (1)

(57) [Claims] 1. A method for measuring the activity of diaphorase, comprising the step of measuring the activity of diaphorase using nitroblue tetrazolium, wherein ethylenediaminetetraacetic acid is contained in the measurement solution.