JPS63152995A - How to measure polyamines - Google Patents

Abstract

PURPOSE:To enable accurate and quick determination of a polyamine, by treating a polyamine-containing specimen with oat-originated polyamine oxidase and putrescine oxidase and measuring the quantity of produced hydrogen peroxide. CONSTITUTION:A polyamine-containing specimen such as urine, blood, lymph, etc., is adjusted to 6.5-9.3pH using a buffer solution, etc. The specimen is treated with oat-originated polyamine oxidase and putrescine oxidase to produce hydrogen peroxide of the molar number corresponding to the total guantity of the polyamines in the specimen. The quantity of polyamine can be determined by measuring the quantity of produced hydrogen peroxide. The oat-originated polyamine oxidase can be produced by homogenating sprouted oats together with a sodium chloride solution, separating the oxidase using salting-out, centrifugal separation, etc., and dialyzing with a buffer solution.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for measuring polyamines using an enzymatic method.

[Conventional technology and problems]

Polyamines are non-protein, low molecular weight, aliphatic basic compounds that are widely distributed in nature. They have various physiological activities and play important roles in promoting protein synthesis, expressing enzyme activity, and in the metabolism of nuclear chemicals. It has a deep connection with reactions. In mammalian bodies, putretone, cadaverine, spermidine, and spermine are the main polyamines, and these four types of polyamines are collectively referred to as total polyamines.

In addition, polyamines have a deep relationship with cell proliferation, so
It has been reported that in cancer patients with actively proliferating cancer cells, total polyamines in so-called body fluids such as blood and urine are significantly increased compared to healthy individuals. Therefore, measurement of total polyamine 9 in body fluids is expected to be a powerful diagnostic method for cancer, and to be widely applied in the field of clinical testing, such as determining the effectiveness of cancer treatment and diagnosing prognosis.

Various methods for measuring total polyamines have also been reported. For example, JP-A-56-36918, JP-A-59-
No. 2700, JP-A-58-141798, etc.

Among these, the Tokukai method No. 56-36918 is a method in which amine oxidase derived from germinated soybeans is applied to a sample containing polyamine, and the hydrogen peroxide produced is quantified.
In the method of JP-A-59-2700, ascorbic acid oxidase is applied to a polyamine-containing sample in advance to remove reducing substances, and then amine oxidase derived from germinated soybeans is applied to quantify the produced hydrogen peroxide. It's a method. However, both of these total polyamine selection methods have the following drawbacks. In other words, the amine oxidase derived from germinated soybeans used to oxidize polyamines is
Putrescine, cadaverine, and spermidine produce 1 mole of hydrogen peroxide, but spermine produces 2 moles of hydrogen peroxide, so hydrogen peroxide production does not correspond to the total number of polyamine moles in the sample. Therefore, the total polyamine content determined by these methods will have a positive error from the true value. Therefore, when a sample containing a large amount of spermine, such as blood, is used as a sample, these methods for measuring total polyamines have large errors and cannot be used.

On the other hand, the method of JP-A-58-141798 is one of the excellent polyamine analysis methods that uses Micrococcus flavidus putrescine oxidase to oxidize polyamines. However, the substrate specificity of putrescine is weak for spermine among all polyamines, and tends to be unsatisfactory when a sample containing a large amount of spermine, such as blood, is used as a sample.

In addition, micrococcycetes were added to samples containing polyamines.
A method for measuring total polyamines has also been proposed by using a combination of putrescine oxidase from Roseus and polyamine oxidase from corn. Although this method is an excellent method for measuring polyamines, corn-derived polyamine oxidase has a relatively large Michaelis constant (Ckj value) for spermine, and tends to have low enzyme productivity, and is somewhat unstable, making it difficult to purify noodles. be. Therefore, the measurement time is long and the measurement sensitivity is low, sometimes resulting in lack of accuracy, and the results have not yet been fully satisfied.

[Means for solving problems]

In view of this situation, the present inventors conducted research in order to develop a method for economically, quickly and accurately measuring the total amount of borea without requiring complicated operations. As a result, the polyamine oxidase derived from oat has a very small value for polyamines, has high productivity, and by combining the polyamine oxidase and putrescine oxidase and reacting them in a certain pH range, both polyamine oxidation molecules can be oxidized. The present invention was completed based on the knowledge that the present invention acts accurately and quickly on polyamines.

That is, the present invention allows polyamine oxidase to act on a polyamine-containing sample and determines the amount of free polyamine in the sample by quantifying the generated hydrogen peroxide.
As the polyamine oxidase, oat-derived polyamine oxidase and Putrescine bovine cedar-v4-
PR&5~9.3 (1) A method for measuring polyamines, characterized by using a cell that operates in range B.

In the present invention, samples containing polyamines include urine, blood, lymph fluid, ascites, and fresh water.

All body fluids such as semen and saliva can be used, as well as microbial culture fluids and animal and plant tissues. Also, if the sample contains measurement-inhibiting substances, such as colored substances or reducing substances, the sample should be subjected to appropriate pretreatment! It is preferable to use t anywhere. The pretreatment method varies depending on the sample, but it involves extraction of polyamines using ion exchange resin or silica gel.

Extraction of polyamines with organic solvents, protein removal treatment with trichloroacetic acid or perchloric acid, etc. are used as appropriate. For samples containing non-free polyamines such as acetyl complex polyamines, such as urine samples,
It is preferable to perform a hydrolysis treatment using standard #A alcohol or an acylpolyamine hydrolase, etc., to convert all the polyamines into free polyamines, and then prepare the sample for this measurement.

Furthermore, the polyamine oxidase used in the present invention must be derived from oats! There is. Oxidase is contained in large amounts in germinated oats.

(approximately 1O unit/gram bud) The extraction method from the oats is not particularly limited, but it can generally be prepared, for example, by the following method. For example, oat sprouts are homogenized by adding 0.1 M sodium chloride solution, and ammonium sulfate is added to the filtrate, which is filtered through gauze or the like, for salting out. Collect the precipitate by centrifugation, and add 0.05M-Quen Chen buffer (PHa, O) to this precipitate. In addition, after dissolving the precipitate, it is dialyzed against the same buffer. Even this level of partially purified enzyme is sufficient for practical use.

Of course, it may be re-purified and used if necessary.

Yet another polyamine oxidase for use in the present invention is putrescine oxidase. The putrescine oxidase may be any type of putrescine oxidase as long as it reacts with at least putrescine and cadaverine out of the total polyamines and produces 1 mole of hydrogen peroxide each when oxidizing the reactive polyamines. Examples of those that are generally suitably used include Micrococcus 7 rabidus putrescine oxidase and Micrococcus roseus putrescine oxidase. These putrescine oxidases may be prepared from within the microorganism 11 using known biochemical techniques.

In the present invention, polyamine oxidase derived from oat is used as the polyamine oxidase,
Furthermore, it is necessary to use the polyamine oxidase in combination with putrescine oxidase. However, it is still unclear what function or factor of oat-derived polyamine oxidase interacts with putrescine oxidase to exert the effects of the present invention.

In the present invention, the polyamine-producing enzyme tkP
It is also an important factor that H&5 to 93 preferably be operated in the range of 6.8 to 9.0.

Although the pH may be adjusted by adjusting the pH of the sample, it is generally preferable to use a buffer. The buffer may be of any type as long as it has a buffering effect, and for example, phosphate buffer, Tris buffer, borate buffer, citrate buffer, and a series of Gud buffers are preferably used. It is preferable to adjust the pH using the above buffer before adding polyamine oxidase to the sample whose total polyamines are to be measured, but it is also possible to adjust the pH of the sample at the same time by adding polyamine oxidase dissolved in the buffer. is also possible.

The action temperature of the polyamine oxidase in the present invention is not particularly limited and may be selected and adopted as appropriate, but generally 2
A temperature range of 0°C to 45°C, preferably 30°C to 40°C is suitable.

In the present invention, by the action of the polyamine oxidase, hydrogen peroxide is produced in a mole number corresponding to the total polyamine 1 in the sample. By measuring the generated hydrogen peroxide, the amount of polyamine can be determined. Any known method for quantifying hydrogen peroxide can be applied to quantify the hydrogen peroxide. For example, a suitable method is to carry out oxidative condensation of 4-7 minoantibiline and a chromogen, which is a hydrogen donor, with hydrogen peroxide in the presence of periosidase, and to compare the colors of the produced pigment. In this case, the chromogens include phenol,
2,4-dichlorophenol.

N-Ethyl-N-(2-Hydroxy-3-Sulfopropyl)-Atsushi-Toluidine φ9m, N-Ethyl-'
N-(2-hydro-3-sulfopropyl)-3,5
-Dimethoxyaniline sodium salt and diethylanine are used.

The method of determining the total amount of polyamines in a sample in the present invention is not particularly limited, but can be determined, for example, by the following method. Polyamine standard solution t-1111L of known concentration (Cp), the polyamine standard solution and the sample are reacted in the same operation, the generated hydrogen peroxide is colored according to the above quantitative method, and the absorption & (E*) and sample absorbance (It
) and reagent-blind absorbance (Ek+) according to formula 1.

Formula l 8 days-Eb [Action and Effect] One of the features of the present invention is the combination of putrescine oxidase and oat-derived polyamine oxidase. By acting in combination, 1
By generating hydrogen peroxide in a mole number corresponding to the total amount of polyamine in the sample in the step and quantifying the hydrogen peroxide, the total amount of polyamine in the sample can be determined economically and accurately.

Another feature of the present invention is that a combination of oat-derived polyamine oxidase, which has a very low i value for polyamines, and seven putretsucinol sidases is allowed to act in a specific pH range, resulting in a very rapid sensitization reaction of all polyamines. As mentioned above, the action PR range is PH6.5-93, preferably PHIL8-93.
90, and more preferably a pH of 7.2 to &2. The effect of combining both polyamine-enriched monomers in this pH range will be specifically shown.

Table 1 shows the case where only 12 units of oat-derived polyamine oxidase were allowed to act on putrescine 20 nmol·/− and spermine 20 nmol·/ilj as substrates in α1M Tris-HCl buffer (PH&O) at 30°C. The results shown in Table 1 show the relationship between reaction time and absorbance when only 20 units of putrescine oxidase derived from Micrococcus 7rabidas were used and when both were used together. is shown in Figure 1. In Fig. 1, the curve ■ represents the case where only 12 units of oat-derived polyamine oxidase was applied, and the curve ■ represents the case when only 20 units of butretsushin sidase from Micrococcus flavidus-derived rice were applied.
The curve (2) shows the case where both are applied together.

Table 1 When both are used together, the coloring intensity corresponding to total polyamine II (in this case, putrescine + sperone) is obtained (but when used alone, cedar in putrescine <t-) The entire polyamycin V gelatinization is completed much faster (approximately 7.5 minutes) than the time required for synoxidase to intensify all of the substrate (putrescine) (approximately 15 minutes).

Table 2 shows the results of the same experiment as in Table 1 but with different PR of the reaction solution. The relationship between PH and the time required to complete the reaction (reaction time) is shown.

PR buffer (TEL) For HILO ~ 6.0, use αIM Quen R buffer, for pH & 5 ~ 7.0, use α1M phosphate buffer, and for pH 7,5 ~ 9,0, use αIM phosphate buffer (0.1M)) At pH 9.5 to 1α0, 0.1M aqueous buffer was used. Putrescine oxidase in the table is PUO
, polyamine oxidase 1lkPAo, putrescine is abbreviated as Put, and spermine is abbreviated as Spm.

Table-2 Items that correspond to the book! Ite, PH45~9.3 no 1 lil!
With PI18 to 9.1, oxidation is possible in a short time.
At 0, all polyamines can be intensified within 30 minutes. Furthermore, P
In the range of H7,2 to 1L2, the reaction is completed within 10 minutes, which is about half the time required to oxidize all putrescine when putrescine oxidase acts alone. Of course, this reaction time will vary depending on the particular enzyme lid used.

This phenomenon is probably caused by the fact that putrescine oxidase is inhibited by spermine, a substrate analog, but oat polyamine oxidase is not inhibited by putrescine, a substrate analog. Since the i value for the sample is very small, the spermine in the sample is quickly oxidized and disappears. With the disappearance of spermine, the inhibition of putrescine oxidase by spermine may be alleviated in a short time, and the oxidation reaction of putrescine oxidase may be completed quickly.

Next, the present invention will be explained in more detail in Examples. In the examples, the activities of putrescine bovine sidase, Boria spp. bovine sidase, and peroxidase were determined by the following measuring method.

(1) Putrescine oxidase activity measurement method αI
M) 'JX-jj & buffer solution (PHNO) 100-4
-Amino7 10.2ml of 19,2°4-dichlorophenol and 5η of peroxidase (manufactured by Sigma, type ■) were dissolved to prepare a coloring solution. Coloring liquid α75
- Add 0.15U of 5mM putrescine to 30"C
After drying, 0.051 u of enzyme solution is added and reacted. 510·! for 1 minute after the start of the reaction! The increase in absorbance (ΔA310) in L is measured and the enzyme activity is calculated according to Equation 2.

In this case, the molecular extinction coefficient for color development is g, 600
(f'aa-') is used. Cedar in Butretsushino - 1/1 unit is PH&O.

1 per minute at 30℃! It is Kozosha, which oxidizes putrescine of 1110 engineering.

Formula 2 Putrescine oxidase activity (unit/-) 0.05
9.6 Q) Method for measuring polyamine oxidase activity.

In the method for measuring the activity of putrescine oxidase in (1) above, α] M)! If α1M phosphate buffer (PHa5) is used instead of J-sufunshun buffer (PR&O) and 5 rnM spermine is used instead of 5mM putrescine as a substrate, polyamine OJ
? 9dase activity is determined.

(3) Method for measuring peroxidase activity Guaiacol α22- is dissolved in 100-g purified water to prepare a guaiacol reagent. 30-width hydrogen peroxide solution α083-100- is dissolved in purified water to obtain a hydrogen peroxide reagent.

α】M phosphate buffer (PE7.O) Add guaiacol reagent Q, 05- and hydrogen peroxide reagent α20- to M phosphate buffer (PE7.O), incubate at 30°C for 3 minutes, and then add enzyme solution 0.05-. The increase in absorbance at 436 nm (ΔA436) for 1 minute after the start of the reaction is measured, and the enzyme activity is calculated according to Equation 3.

Formula 3 Peroxidase Activity (Units/-) The abbreviations for reagents used in the following examples are as defined by the sludge.

Standard solution: 3 Q nmol/- putrescine aqueous solution.

Buffer A: α4M) Lis-hydrochloric acid buffer (PH&O) Enzyme reagent A trαIM phosphate buffer (PH7,2) containing 30 units of Niacylpolyamine hydrolase.

Hemolysis t&: 1.5%)9. ): /X-100 included
rαIM tris-salt mat solution (PHILO).

Column A: 1ill! I want to fill it with cation exchange resin α12JI.

Desorption solution A: (14M)'l chloroacetic acid solution.

Desorption solution B: α4M catalytic solution.

Neutralizing solution: α3M) lis-(hydroboxydimethyl)-aminomethane solution.

Enzyme test 1 & B: Putrescine sidase (Micrococcus no. Flavidus Shirarai) 20 units/Hl, polyamine sidase (derived from oat) α2
Units/-, Peruo Bovine Sidase (manufactured by Sigma, Type ■) 10 Units/-04-7 Minoantibilin 0.3 ■/m, 2.4-dichlorophenol 15q/-
containing bαIM) Lis-HCl buffer (PH&O) Furthermore, in the following examples, putrescine is
Cadaverine is abbreviated as Cad, spell scolding gin is abbreviated as 5P (l), and spermine is abbreviated as Spm.

Example 1 4-aminoantipyrine 1α2η, 2.4-garipmaphenol 4.9%, peroxidase 33G units (L
IM) It was dissolved in a lysate-salt buffer solution (PH 7, 8) and used as a coloring reagent.

Color reagent A! 8211j with 2mMptm as a substrate solution.
t or Q IBM (a A or 2111M 811
'! or 2 mM Sp m or a 1:1:1!1 mixture of these four types of polyamine solutions to α08-
Add and incubate at 37°C for 3 minutes.

After incubation, either the enzyme 20 units of putrescine oxidase derived from Mikukotsucus flavidus, only 12 units of polyamine oxidase derived from oat, or both 20 units of putrescine oxidase and 12 units of polyamine oxidase derived from oat was added. (all amounts added were α1-), the mixture was reacted at 37°C for 10 minutes, and 510! The absorbance at 100 mm was measured.

The results are shown in Table-3.

Table 3 According to experiment numbers 1 to 5, this putretsucinol sidase was
It can be seen that equimolar amounts of hydrogen peroxide are produced. α0
An absorbance of 10 corresponds to reagent blinding. P in experiment number 5
U t e Ca 6 and 5) The absorbance (approximately α61) corresponding to the total gills of d was not obtained because of s 8 P.
This is because the reaction has not yet been completed due to inhibition of ``. Experimental pots 6 to 10 show that oat-derived polyamine oxidase uses 81111 and 5pat-substrates and produces equimolar amounts of hydrogen peroxide. From Experiment Nos. 11 to 15, it can be seen that by combining both enzymes, the total amount of polyamines can be measured accurately and quickly in polyamine samples of any composition.

Example 2 Essence water, standard solution, urine, put of each I L 5 nmol*7-@Caae 5 pat all (added urine 1), put of 240 gaole/- each.

Ca Ae Sp am S Xl”k strain Ca Lf urine (
Five samples of added urine 2) were used as samples, and the total amount of polyamines was measured according to the following measurement procedure.

[Measurement operation]

(1) Add buffer ALO- and enzyme reagent A to sample 1.0-
1. After stirring OmV'a1JaL, the mixture was kept at 37° C. for 1 hour to liberate acetyl polyamine.

(2) Centrifuge at 3,000 r) pm for 5 minutes,
Pour the entire amount of supernatant liquid into a column and allow the polyamine to be adsorbed onto the ion exchange resin.

(3) Wash the column with purified water 101114.

(4) Elute the polyamine with desorption liquid A1.0-, and neutralize it by adding neutralizing liquid A1.0-. (PH will be approximately &0) (5) Add 1.0 - of # elemental reagent B, keep warm at 37°C for 10 minutes, and measure the absorbance V at 510 am using purified water as a control.
Measure.

The luminous intensities of the five samples based on this measurement procedure were as shown in Table 4. In addition, the polyamine value calculated using the above-mentioned formula 1 is also shown.

Table 4 Calculating the spiked recovery rate from the values in the table, spiked urine 1 is 97
．． 6%, and added urine 2 was good at 9&3%, indicating that the amount of polyamines in urine was measured correctly.

Implementation-Example 3 Purified water, standard solution, blood, 1 each! 5 mmole/kj
put, Caa, 5pds SpmY added blood (
Added blood 1), each 2 & Onmole/kj Put
Five types of samples of blood (added blood 2) to which s (aa, 1 pat 8 pym') had been added were used to measure the total amount of polyamines according to the following measurement procedure.

Ten blood samples of each of the three types were measured and a simultaneous relapse test was conducted.

[Measurement operation]

0) Add hemolysate 10- to sample 1.0- to lyse red blood cells.

13 Centrifuge at 3.000 rp for 5 minutes, pour the entire supernatant into a column, and ion exchange the polyamine II! Adsorb to resin.

O) Wash the column At' twice with purified water λ0IIJX.

Elute the polyamine with desorption liquid B1.011j, and neutralize it by adding neutralizing liquid 10-. (PH will be approximately &0) (5) Add Kenso test 4B&to-, keep warm at 37°C for 10 minutes, and then measure the luminous intensity at 510 nw* using purified water as a control.

Table 5 shows the results of the simultaneous reproducibility test.

Table-5 The simultaneous reproducibility is very good, less than 2 times.

It can be seen that the amount of polyamines in blood can be measured with high accuracy by this measurement method.

Table 6 shows the absorbance and polyamine values of the five samples. For blood samples, the average value of 10 samples was adopted.

Table 6 Calculating the spike recovery rate from the values in the table, the spiked blood 1 is 9
9.4, Added Blood 2 had a very good value of 10α9≦, which shows that the amount of polyamine in blood can be accurately measured by this measurement method.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between the reaction time and the change in absorbance based on the hydrogen peroxide produced when a system containing putrescine and spermine is made to react with oat-derived polyamine and cedar. Similarly, curve (2) shows the graph when only sidase in putretsucinol was allowed to act, and curve (2) shows the graph when both enzymes were made to act together.

Claims (1)

[Scope of Claims] When determining the amount of free polyamine in a sample by acting a polyamine oxidase on a sample containing a polyamine and quantifying the hydrogen peroxide produced, oat-derived polyamine oxidase is used as the polyamine oxidase. and putrescine oxidase at pH 6.
A method for measuring a polyamine, characterized in that it acts in a range of 5 to 9.3.