JPS6338198B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for quantifying the concentration of acetylputretscine by causing acetylputretscine oxidase to act on a system containing acetylputretscine and measuring the amount of hydrogen peroxide generated. It is related to.

Polyamines such as putrescine H ₂ N
( _CH2 ) _{4NH2 ,} spermidine _H2N ( _CH2 ) _4NH
( _CH2 ) _{3NH2 ,} spermine _H2N ( _CH2 ) _3NH
(CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ etc. are found in animals,
It is widely distributed throughout the living world as a component of all living cells such as plants and bacteria, and is known to be involved in cell division, proliferation, and the metabolism of nucleic acids, which form the biochemical background.

By the way, in recent years it has been reported that the amount of polyamines in the urine of cancer patients is higher than that of normal people.
Research (Chancer Rosearch), Volume 31, No.
[pages 1555-1558], many researchers have conducted research on the relationship between the amount of polyamines in so-called body fluids such as urine, blood, and lymph and cancer diseases. It has become clear that fluctuations in polyamine excretion reflect changes in the disease state of cancer patients, and the quantification of polyamines has become important in clinically diagnosing cancer and understanding the state of cancer cure. Ta.

Conventionally, methods for quantifying polyamines include gas chromatography (Clinical Chemistry, Vol. 19, No. 904-
907 pages], Amino Acid Analyzer Method (FEBS Letters, Vol. 46, No.
Pages 305-307], High Performance Liquid Chromatography Method (J. Chromatography, Vol. 145, No. 141)
However, these chemical methods lack rapidity and require additional processing after hydrolysis with acids and before converting polyamines into measurable derivatives. This method has many practical inconveniences, such as requiring processing and making operations complicated.

On the other hand, a method for quantifying free polyamines using the specific action of an enzyme (Japanese Patent Publication No. 56-36918), a method for quantifying spermidine and spermine (Japanese Patent Publication No. 56-21398), spermine, spermidine, and putretsucine. Method for quantifying
50-9492) have been proposed. Unlike chemical methods, these enzymatic methods do not require special pretreatment to make derivatives, and are preferred methods as they are considerably improved in terms of rapidity. Since the purpose is to quantify polyamines, if acetyl polyamines are present, they must be hydrolyzed with hydrochloric acid or deacetylated with enzymes to convert them to free polyamines, which makes the operation difficult. In addition to being complicated, acetyl polyamines cannot be directly quantified.

However, according to recent research, the majority of polyamines in human urine are acetylputretscine, and it is becoming clear that this increase or decrease is effective in diagnosing cancer and determining the status of cancer cure. There has been a wide demand in this field for a method that can more easily quantify acetylputretscine.

In view of these circumstances, the present inventors have conducted intensive research to develop a method that can quickly, easily, and accurately quantify acetylputretscine. It was discovered that this object could be achieved by allowing synoxidase to act and measuring the concentration of hydrogen peroxide produced, and based on this knowledge, the present invention was accomplished.

That is, the present invention quantifies the concentration of acetylputretscine by causing a novel enzyme, acetylputretscine oxidase, to act on a system containing acetylputretscine and measuring the concentration of hydrogen peroxide produced. The present invention provides a method.

Acetylputretscine oxidase is produced, for example, by Aspergillus oryzae.
This is a novel enzyme produced by IAM2682 that has not been described in any literature, and can be produced by the method described below. Aspergillus oryzae IAM2682 (obtained from the Institute of Applied Microbiology, University of Tokyo) was cultured with a medium composition of NaNO ₃ 0.1%,
Contact with a medium consisting of KH ₂ PO ₄ 0.1%, MgSO ₄ 0.05%, KC 10.05%, glucose 3.0 and culture at 28°C for 48 hours, and the seed culture thus obtained was
0.1%, _{KH2PO4 0.1} %, _{K2HPO4 0.15} %,
Main culture was carried out for 48 hours in addition to a medium consisting of 0.02% MgSO ₄ and 0.2% acetylputretscine. After culturing, the bacterial cells were collected by filtration, washed with 0.01M phosphate buffer (PH6.8), suspended in the same buffer,
Destroyed it with a dyno mill. A supernatant was obtained from this disrupted solution by centrifugation at 7000 rpm for 20 minutes, and the extract was immediately collected.
Buffered with 0.01M phosphate buffer (PH6.8)
Pass through DEAE and cellulose to adsorb acetylputrescine oxidase, then wash with 0.1M phosphate buffer (PH6.8) and elute using a linear concentration gradient method. The eluted active fraction is subjected to ammonium sulfate fractionation at an ammonium sulfate concentration of 50% to 90% saturation, and then dialyzed against 0.01M phosphate buffer (PH6.0). Next, the dialyzed enzyme solution was passed through a hydroxyapatite column buffered with the same buffer solution, and the active fraction was adsorbed.
Wash with 0.01M phosphate buffer (PH6.0), and then
The active fraction is eluted using a linear concentration gradient method using 0.01-0.2M phosphate buffer (PH6.0). The eluent
After concentration with 90% saturated ammonium sulfate, Sephadex G-
Purified enzyme was obtained by separation using 200 ml. This purified enzyme has the following physical and chemical properties.

(1) Action: Acts on acetylputrescine to produce 1 mole of 4-acetylaminobutanal and 1 mole of hydrogen peroxide.

(2) Substrate specificity: acts on acetylputretscine, but not on spermine, spermine, putretscine, N-acetylspermine, ^N1 -acetylspermidine, and ^N8 -acetylspermidine.

(3) Optimum pH; around 8.

(4) PH stability: At pH 6.0 to 8.5, it has a residual activity of 85% or more even after heat treatment at 37°C for 30 minutes.

(5) Optimum temperature: around 50℃ at PH7.5.

(6) Temperature stability: 90% residual activity after treatment at 40°C for 30 minutes at pH 7.0.

(7) This enzyme contains copper.

(8) Isoelectric point; around 5.0.

The mechanism of action of this enzyme on the substrate is shown in the reaction formula as follows.

Decomposition reaction formula of acetylpolyamine by acetylputrescine oxidase Acetylputrescine oxidase acts only on acetylputrescine among acetylpolyamines, producing equimolar amounts of ammonia and equimolar amounts of hydrogen peroxide, but does not act on other acetylpolyamines or free polyamines. .

The method of the present invention directly determines the concentration of acetylputrescine present in acetylpolyamines or a mixed system of acetylpolyamines and free polyamines, which was completely impossible using conventional methods.

Next, preferred embodiments of the present invention will be described. Note that the enzyme unit shown here is defined as one unit having the amount of enzyme required to produce 1 μmol of hydrogen peroxide per minute, which was measured as follows.

Measurement of acetylputretscine oxidase; 0.5ml of 10mM acetylputretscine as substrate
The enzyme activity is determined in the same manner as in the reference example except that 0.1M phosphate buffer with pH 7.5 is used as the buffer for the coloring solution.

In a preferred embodiment of the method of the present invention, 0.05 to 0.5 units of acetylputrescine oxidase is first added to a sample containing acetylputretscine to oxidize the acetylputrescine. Assuming that the amount of hydrogen peroxide produced in this reaction step is f moles, the amount is expressed as follows.

f (moles) = (number of moles of acetylputretsucine) Therefore, the concentration of acetylputretsucine is Acetylputretsucine (μM) = K・f However, K = 1 × 10R ₆ /molecular extinction coefficient Method of the present invention To measure the amount of hydrogen peroxide in a 4-aminoantipyrine/phenol/peroxidase system or 4-aminoantipyrine/
In addition to the colorimetric method using an N,N-diethylaniline/peroxidase-based reagent, it is also possible to use a fluorescence method, a luminescence method, etc., which are commonly used for quantifying hydrogen peroxide. Furthermore, instead of measuring the amount of hydrogen peroxide, it is also possible to measure the corresponding amount of decrease in oxygen or increase in ammonia or aldehyde.

According to the method of the present invention, when measuring acetylputrethcine as in the past, there is no need to hydrolyze acetylputrethcine with hydrochloric acid or enzymes, and special methods such as high-performance liquid chromatography and amino acid analyzers are not required. Even chemical methods using modern equipment were considered extremely difficult. Because acetylputretscine can be measured quickly, easily, and accurately,
The method of the present invention is very useful for diagnosis and testing of cancer and other diseases.

Next, the present invention will be explained in more detail with reference to Reference Examples and Examples.

Reference example: peroxidase 5mg, phenol 0.2ml, 4
- Prepare a coloring solution by dissolving 10 mg of amino-antipyrine in 0.1M phosphate buffer at pH 8.0, and perform the operation using acetylputretscine as a substrate and about 0.5 units of acetylputrescine oxidase as an enzyme. Ivy. The results are shown in graphs in FIGS. 1 and 2. As is clear from this graph, the reaction is completed within about 5 minutes, and 1 mole of hydrogen peroxide is produced from 1 mole of acetylputrescine.

In addition, the various acetyl polyamines used in the reference examples and the following examples are "Methods in Enzymology",
17B, pages 829-833 from free polyamines.

Example 1 Similarly to the reference example, 5 mg of peroxidase, 0.2 ml of phenol, and 10 mg of 4-aminoantipyrine were added to PH.
A coloring solution was prepared by dissolving it in 100 ml of 0.1M phosphate buffer (8.0).

1.5ml of this coloring liquid, spermine, spermidine,
2.0 mM each of putretzcine, acetylspermine, N ¹ -acetylspermidine, N ⁸ -acetylspermidine and acetyl putretzine
3 volumes of 25 μ each of the prepared aqueous solution and 0.82 ml of distilled water
into a ml cell. The concentration of each polyamine component in this solution was 20 μM.

After preheating the solution prepared in this manner at a temperature of 35° C. for 3 minutes, 5 μ (about 0.5 units) of acetylputrescine oxidase was added to the solution, and after reacting for 10 minutes, the absorbance at 505 nm was measured.

Based on this value, the molecular extinction coefficient of this coloring solution is
The acetylputrescine concentration was calculated from the formula for calculating each component in the first embodiment using the constant K in which 6250 was substituted and found to be 19.7 μM.

This value is in excellent agreement with the added concentration value of 20.0 μM, and it can be understood that the method of the present invention has high reliability.

Example 2 Peroxidase (Boehringer grade) 5.0 mg, 4-aminoantipyrine 10.0 mg, sodium-N-ethyl-N-(2-hydroxy-3
-sulfopropyl)-m-toluidine 54.8mg
3 ml of 1.5 ml of coloring solution prepared by dissolving in 100 ml of 0.1M phosphate buffer (PH8.0), 0.5 ml of urine, and 0.5 ml of distilled water.
The mixture was injected into a cell, and after preheating at 35°C for 3 minutes, 5μ (approximately 0.5 unit) of acetylputrescine oxidase was added, and after reacting for 15 minutes, the absorbance at 555 nm was measured.

The amount of change in absorbance obtained by this reaction was as follows.

Absorbance change amount g = 0.035 Using this value and the molecular extinction coefficient of 17200 based on this color development, and substituting it into the component calculation formula of the above embodiment, the concentration of acetylputretsucine in the reaction solution (2.5 ml) is calculated. It was calculated as follows.

Acetylputretscine = 1.74 μM The acetylputretscine concentration in urine water converted from this value is as follows.

Acetylputrescine = 8.7 μM Example 3 Add spermine, spermidine, putretzine, N ¹ -acetylspermidine, N ⁸ -acetylspermidine, acetylputrescine to 1.5 ml of a coloring solution prepared by dissolving the same components as in Example 1. each of
50 μM each of a 2.0 mM aqueous solution (the final concentration of each of these known components is 40 μM), 0.25 ml of control serum (1 bottle of Corn Sera "Nitsui" dissolved in 1.5 ml of distilled water), and distilled water. 0.45ml 3
ml cell and preheated at 35°C for 3 minutes. Next, 5μ (approximately 0.5 units) of acetylputretscine oxidase was added, and after reacting for 10 minutes, the absorbance was measured at 505nm.The result was calculated by substituting the measured value of the absorbance change.Acetylputretscine: 39.2μM
It was hot.

The concentration of acetylputretscine measured by the quantitative method of the present invention was 40 μM at each component concentration.
It is confirmed that the method of the present invention has excellent practical value.

Example 4 A coloring solution with the same composition as the reference example was prepared, and 1.5ml of this coloring solution, 100μ of each 2.0mM aqueous solution of acetylputrethscine (final component concentration 80μM), and 0.90ml of distilled water were placed in a 3ml cell. After preheating at 35℃ for 3 minutes, add 5μ of acetylputrescine oxidase.
(approximately 0.5 unit) was added and reacted for approximately 10 minutes, and the absorbance based on hydrogen peroxide produced by oxidation of acetylputretscine was measured. The change in absorbance at 505 nm of the step obtained in this reaction was 0.528. This value was 81.9 μM for acetylputretscine, which is in good agreement with the concentration of the prepared components.

[Brief explanation of drawings]

Figures 1 and 2 show the relationship between the absorbance that changes based on the time and hydrogen peroxide produced when each acetylputretscine is oxidized using acetylputretscine oxidase. This is a graph showing.

Claims (1)

[Claims] 1. An acetylputretscine characterized by allowing acetylputretscine oxidase to act on a system containing acetylputretscine, measuring the amount of hydrogen peroxide produced, and quantifying the concentration of acetylputretscine based on this. Method for quantifying syn.