JPS6236199A - Method of determining calcium ion - Google Patents

Abstract

PURPOSE:To determine a calcium ion, simply by reacting a calcium ion with calmodulin to form a calcium calmodulin complex, activating a calmodulin- depending enzyme with the complex and measuring the enzymatic activity. CONSTITUTION:A fixed amount of a calcium ion-containing test specimen is blended and reacted with a calmodulin-containing buffer solution. The reaction solution is reacted with a calmodulin depending enzyme and its substrate and a ratio of change of the reaction product to be detected is determined. The calcium ion corresponding to the ratio of the change can be obtained from a preobtained measuring.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for quantifying calcium ions, and more specifically to forming a calcium-calmodulin complex, thereby activating a calmodulin-dependent enzyme, and measuring its activity. This invention relates to a method for quantifying calcium ions.

Conventional technology and problems Calcium is a metal in living organisms and is present in bones, teeth, and hard tissues, but it is present in serum at 10 mg/clj! Around 4 to 12 μ/a is contained in urine. Calcium ions are intracellular information transmitters that are involved in nerve transmission, muscle contraction,
It regulates the relaxation and secretion systems. Therefore, quantitative determination of calcium ions is useful for diagnosing hyperparathyroidism, vitamin D deficiency, renal failure, and the like. Although the concentration of calcium ions in blood or urine is relatively high, conventional measurement methods are complicated to operate. In other words, the chemical colorimetric method (Orthocr) is used for the measurement of calcium ions.
Connery.

)1. V, & Br1gg5. ^、R、; Am
,J,Cl1n,Path,.

45, 290.1966) Atomic absorption spectrometry (Zeltn
er, A, &Se11g5on, D, : C11
n, Chemo, 10, 869.1964) ion electrode method (Arnold, B.: Amer, J.
Cl1n.

Path, , 49. 627.1968) are known, but all of them are complicated to operate and easily affected by other substances.

Means for Solving the Problems According to the present invention, calcium ions in a sample and the protein calmodulin are combined to form a calcium-calmodulin complex, and this complex activates a calmodulin-dependent enzyme. Calcium ions can be quantified by measuring activity.

The principle of the invention is as follows.

Calcium ions react with inactive calmodulin to form active calcium-calmodulin complexes. When an inactive calmodulin-dependent enzyme such as phosphogesterase acts on this complex, this enzyme is activated. Calcium ions in a sample can be quantified by measuring the enzyme activity of this activated enzyme. When carrying out the method of the present invention, first, a certain amount of a sample containing calcilum ions and a calmodulin-containing buffer are mixed and reacted. A calmodulin-dependent enzyme and its substrate are added to the reaction solution and reacted, and the rate of detectable change in the reaction product is quantified. Calcium ions corresponding to the rate of change can be determined from a previously determined calibration curve.

Examples of samples containing calcium ions include normal blood (particularly serum) and urine.

Calmodulin is extracted from mammalian tissues, plants, protozoa, coelenterates, microorganisms such as Eurospora crap, Escherichia coli, etc., but a suitable example is one derived from cow brain. . After crushing the bovine cerebral cortex, the supernatant of the centrifugation is applied to a DEAE-cellulose column and eluted using a concentration gradient method, whereby calmodulin and calmodulin-dependent phosphogesterase are separated and obtained. By further purifying these using common protein purification methods, highly purified calmodulin and calmodulin-dependent phosphogesterase can be obtained.

The ratio of the calmodulin-containing buffer solution to the calcium-containing sample is determined to be an appropriate amount for each sample within a range that allows accurate detection operations. For example, if the sample is serum, dilute it 100 to 5000 times with a buffer solution, and
nl of calmodulin-containing buffer (14-1 ml)
(contains 0.1 to 50 u of calmodulin).

The mixed solution of calcium ions and calmodulin is 5~
A calmodulin-calcium complex solution can be obtained by incubating at 50° C. and pH 4 to 10 for 30 seconds to 10 minutes.

As the buffer to be used, any of the examples used in the enzymatic reaction described below can be used.

The calmodulin-dependent enzyme may be any enzyme as long as it is activated by the calcium-calmodulin complex, such as phosphogesterase, adenylate cyclase, NAD kinase, protein kinase, tryptophan hydroxylase, ATPase, Protein kinases and the like are used. An example of a suitable enzyme is phosphogesterase obtained from bovine brain.

All of these calmodulin-dependent enzymes are known enzymes, and their corresponding substrates are also known, so it is sufficient to use a substrate that is compatible with the enzyme used.

Changes that can be detected by performing these enzymatic reactions in the presence of calcium-calmodulin conjugate include a decrease in substrate or product. When these changes can be directly quantified, they may be quantified according to known methods.

When a product cannot be directly quantified, it is desirable to convert it into a compound that can be easily quantified.

For example, when the product is hydrogen peroxide, or when hydrogen peroxide can be produced stoichiometrically by the action of an oxidase that decomposes the product, hydrogen peroxide is converted into peroxidase in the presence of a chromogen. The product is converted into a dye by the action of , and the product can be quantified by measuring the absorption due to coloring of the reaction solution by the produced dye at the maximum absorption wavelength of the dye, and as a result, the calcium ion can be quantified.

Although these reactions may be carried out sequentially, it is also preferable to carry out the reaction in one step by adding a substrate necessary for the enzyme reaction, an enzyme, and a chromogen, and then measuring the absorption of the reaction solution.

Many methods other than those described above are known for quantifying the product, and these methods can be used in combination as appropriate.

When carrying out an enzymatic reaction in one step, a buffer solution containing enzymes, substrates, 9 chromogens, etc. necessary for the enzymatic reaction is prepared as a reagent solution. Each component should be contained in a sufficient amount to carry out the reaction, but usually 0.0 l of each enzyme is contained in 1 ml of reagent.
A reagent solution containing 10 U, 0.1 to 10 μmol of substrate, and a product quantification system is prepared.

The product quantification system consists of a composition containing a necessary amount of an enzyme, a chromogen, etc., depending on the method of quantification of the product, usually in a considerably excessive amount.

These and 1 to 25 times the volume of the reagent solution are added to the above-mentioned calcium-calmodulin complex solution.

The reaction is carried out at 5 to 50°C and pH 4 to 10 for 30 seconds to 60 minutes. After the reaction, the product is quantified according to the selected product quantification system.

As an example, an enzyme reaction system using phosphogesterase as a calmodulin-dependent enzyme and cAMP as its substrate will be described in detail. cAM
P is converted to 5'-AMP by phosphogesterase, and as a method for quantifying this 5'-AMP, 5'
- A method in which AMP is converted into adenosine and phosphate using 5'-nucleotedase and the phosphate is measured chemically or enzymatically, a method in which adenone is oxidized with nucleondokindase and measured, 5'-/IIP is converted into adenylate Kinase, pyruvate kinase, phosphoenolpyrivic acid (PEP),
A method of measuring hydrogen peroxide produced using pyruvate oxidase, a method of measuring pyruvate by the amount of change in NADH using lactate dehydrogenase, 5'-AMP
Examples include a method in which hydrogen peroxide produced is measured by reacting with adenine phosphoryl transferase, adenine deaminase, or xanthine oxidase.

The enzymatic reaction described above is represented by the following formula.

Activation of phosphogesterase Ca2+-calmodillin (active type) + phosphogesterase (inactive type) Phosphogesterase (active (ft[>5'-/Production of MP Quantification of 5'-AMP Dase■5'-AMP + H,O adenosine +
Phosphoric acid11. acid. b) 'i"t "Hiiragi 4 ±-Hayashi ei + 'j'rf'r. 11g-x-1-'Jyya↓ Detection 2 (acetyl phosphate) +2CO2+28202 detection↓ Detection then in the case of ■ above The specific operation will be explained using as an example.First, phospho-gesterase and cAMP.

5'-nucleotidase, purine nucleoside phosphorylase, xaneyoxidase, inosine, system for quantifying hydrogen peroxide produced, e.g. peroxidase to lead to dye, electron donor, phenolic compound and if necessary metal ion, surfactant Prepare a reagent solution containing a buffer solution, etc. The concentration of each component in the reagent solution is phosphogesterase 1-100 mu/mL cAMP
o, 1-5 μmol 7-I, 5'-nucleotidase 0.1-20u 7-I, purine nucleoside phosphorylase 1-100 mu/ml, xanthine oxidase 1-100 mu/ml, inosif 0.1-I Q μm
0.1 to 5 μmol of Mg necessary for the reaction of ol, phosphodi, lr-sterase 0.1 to 20 μmol of Mn required for the expression of 7 to I, 5'-nucleotidase activity
l 7-I, peroxidase 1-100 u/ml,
Electron donor Q, 1 to 1 Oa+ mol/1, and phenolic compound 0.1 to 100 m mo+/β are suitable, respectively. As electron donors, 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolohydratine (MBTH), 2-hydrazinobenzothiazole (
HBT), 2-aminobenzothiazole (ABI), etc. are used. As a phenolic compound, phenol,
N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine (EMAE), 3.5-xylenone N, N-dimethylaniline, diethylaniline,
0-coumarin e, 8-aminoquinolinic acid, 1-amino-
8-naphthol-2,4-disulfonic acid (S
S acid), N (β-hydroxy-γ-sulfoprovir) = 3.5-diethylaniline (H3DΔ), etc. As mentioned above, the metal ions include Mg", Mn
” is required, and compounds that provide these ions include magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, etc. As the surfactant, Triton X-100 and the like are used.

As the buffer, phosphate buffer, imidazole buffer, Tris buffer, succinate buffer, citrate buffer, borate buffer, acetate buffer, glycylglycine buffer, etc. are used. The concentration of the buffer in the reagent solution is preferably 0.01 to 1 mol/β.

Calcium-calmodulin complex solution and 1
~25 times the volume of the reagent solution is mixed, and the enzymatic reaction is carried out at 5 to 45°C, preferably 20 to 40°C, and pH 4 to 10 for 30 seconds to 60 minutes.

When using the enzyme reaction described in (■) above, replace the enzyme reaction system in (■) with nucleoside oxidase 0, O1-10.
The operation can be performed in the same manner as in case ① except that u 7mM is used. When using the enzymatic reaction of
~10 μmol/ml.

A reagent solution containing P E P O, 1 to 10 μmol 7 mM, phosphoric acid 0.1 to 10 μmol 7 mM, peroxidase, an electron donor, and a phenolic compound is prepared and reacted in the same manner as in the case (2). In the above, the concentrations of peroxidase, electron donor, and phenolic compounds are ■
The same method as in case (2) may be used, and the same electron donor and phenol compound as in case (2) can be used. When using the enzymatic reaction (2), adenine phosphoribosyltransferase, adenine deaminase and xanthine oxidase are added at 0. O1-10u/ml, pyrophosphoric acid 0.
1-10 μmol 7mM.

Prepare a reagent solution containing peroxidase, an electron donor, and a phenolic compound, and react in the same manner as in case ①. In the above, the concentrations of peroxidase, electron donor, and phenolic compound may be the same as in case ①, and the concentrations of peroxidase, electron donor,
As the phenolic compound, the same compounds as in the case (2) can be used.

After the enzyme reaction, measure the visible absorption of the reaction solution from 400 to 780 nm.
, preferably in the wavelength range of 500 to 600 nm using a spectrophotometer or the like, and quantify calcium ions in the sample using a calibration curve prepared in advance by applying the above series of operations to a solution containing Ca'' ions at a constant concentration. do.

Aspects of the present invention will be explained below using examples.

Example 1 A calibration curve for calcium ions is determined using bovine brain calmodulin and bovine brain calmodulin-dependent phosphogesterase.

10 Jl containing 0-10 μM calcium ions!
Add 10 ml of 0.20#l lumodiniline solution to 1 ml of sample and react at 37°C for 5 minutes. This reaction solution (20d)
A reagent solution and water (total of 2980 JI
After adding i) and incubating at 37°C for 5 minutes, the absorption of the reaction solution at 500 nm was measured to obtain a calibration curve.

A linear proportional relationship is obtained in the range of 2-10 μM calcium ions.

Δ) Degrading 5'-AMP with 5'-nucleotidase,
The generated phosphoric acid is measured using an enzymatic method.

Reagent solution phosphogesterase 12mM 10 relatives 10mM
Mg5O< ・5820 50 ”6
0mM uni25Q/1
5'-nucleotidase 10mM 10 〃10
mM inosine 40〃Brine nucleoside phosphoryl 10mM
10//xanthine oxidase
10mM 10 Peroxidase (2mg/ml) 100 42mM
Phenol 500 2.4d4-aminoantipyrine 500 Phosphate buffer (I) 87
．． 5) 1000”6mM c-AMP
50 //”20
650 〃Total 2980〃 The calibration curve is shown in FIG.

B) Measuring hydrogen peroxide produced by reacting 5'-AMP with adenylate kinase, pyruvate kinase, and pyruvate oxidase Reagent solution Phosphogesterase 12mM 10 faces 10m
M MgSO4・51(2050II5'-nucleotidase lQmu 1 o 760mM LlnC
j22 50〃Adenylate Kinase 10mM 50〃10mM ATP
1
0 0” pyruvate quina-f
l(1mM 501/10mM
PEP (phosphoenol pyruvic acid)
100 10mM phosphoric acid
100 Pyruvate oxidase
lQmu 5 0 Peroxidase (2■/m+) 100 42mM Phenol 500 2.4mM 4-aminoantipyrine 500 Phosphate buffer (pf17.5)
1000 〃61TIM C-AMP
50 "H2O260" Total 2980 The calibration curve is shown in FIG.

Example 2 Determination of calcium ions in serum Serum was diluted 500 times with phosphate buffer (pH 7.5) and diluted with 10JI! Ten animals of 0.2 U/m calmodulin solution were added to the solution, and after incubation at 37°C for 5 minutes, the mixture was treated in the same manner as in 2) of Example 1, and the calcium ion concentration was determined from the calibration curve. The results are shown in Table 1. EDTA titration method [:J,L
ab, Cl1n, Med, 611029 (1963
) ) The results obtained are shown in Table 1.

Table 1 According to the present invention, calcium ions can be determined quickly and easily.

[Brief explanation of the drawing]

Figures 1 and 2 show calibration curves for calcium ions determined using bovine brain calmodulin and bovine brain calmodulin-dependent phosphogesterase. Figure 1 shows the case where 5'-AMP is degraded with 5'-nucleotidase and the generated phosphoric acid is measured by enzymatic method. This shows the case measured by an enzymatic method using oxidase. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. 1 Prisoner (Ca”) (μ) 2nd mouth [Cα2+] (AM)

Claims (1)

[Claims] Calcium characterized by reacting calcium ions in a sample with calmodulin to form a calcium-calmodulin complex, activating a calmodulin-dependent enzyme with the generated calcium-calmodulin complex, and measuring the enzyme activity. Method for quantifying ions.