JPH10313893A - Measurement of saccharified protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring a saccharified protein, comprising using an enzyme not causing inhibition of reaction even if EDT is included in a specimen when a saccharified protein in a biological sample is measured by fructosylamino acid oxidase. SOLUTION: Fructosylamino acid oxidase which is produced by Fusarium oxysporum S-1F4 (FERM BP-5010) is separated and purified and the purified oxidase is used for measuring saccharified proteins. Thereby, a saccharified protein can be measured without causing reaction inhibition even if EDT is included in a specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring a glycated protein by measuring a glycated protein by an enzymatic method using fructosyl amino acid oxidase. More specifically, a blood sample is collected in a blood collection tube to which EDTA has been added, and even when EDTA is contained in the sample, fructosyl amino acid oxidase capable of measuring glycated protein without reaction inhibition is used. This is an invention related to the measurement method used.

[0002]

2. Description of the Related Art A saccharified protein is a substance produced by non-enzymatically and irreversibly binding an amino group of an amino acid constituting a protein and an aldehyde group of a reducing sugar such as aldose. In addition, since the irreversible bond is called Amadori transfer, it is also called an Amadori compound. The rate of saccharified protein production depends on the concentration, time, and temperature of the protein and reducing sugar.The higher the amount of reducing sugar, the longer the contact time between the protein and reducing sugar, the less denaturation of the protein occurs. The higher the temperature, the faster the rate of saccharified protein production and the greater the production. In a living body, the concentration of a glycated protein varies depending on the half-life of the protein to be glycated, so that various information can be obtained by measuring the concentration of the glycated protein.
For example, the concentration value of glycated hemoglobin in which erythrocyte hemoglobin is glycated or glycated albumin in which plasma albumin is glycated reflects the average blood glucose level in a certain period in the past, so that screening tests in health examinations and diabetes It has been useful as an indicator for diagnosis and treatment of.

Conventional methods for measuring saccharified proteins include a method using high performance liquid chromatography [Chro.
Sci. 10: 659 (1979)], a method using a column packed with a carrier to which boric acid is bound [Clin. Chem. 28: 2088 (198)
2)], electrophoresis [Clin. Chem. 26: 1598 (1980)], method using antigen-antibody reaction [JJCLA 18: 620 (1993), instrument / reagent 16: 33-37 (1993)], etc. Although known, there were problems with the need for expensive dedicated equipment, the accuracy of the measurement results, or the speed with which a large number of samples had to be processed.

[0004] As a method for solving the problems of the prior art, a glycated protein is reacted with fructosyl amino acid oxidase, and the amount of hydrogen peroxide generated by the enzymatic reaction or the amount of consumed oxygen is measured to quantify the glycated protein. There is a way. Further, there is a method in which a glycated protein is digested and fragmented into amino acids or peptides with a protease using fructosyl amino acid oxidase, and then the glycated protein is quantified. (Japanese Unexamined Patent Publication (Kokai) No. 5-192193) These conventional enzymes have not been examined in detail in the state when they are collected as biological samples, for example, the effects of anticoagulants. A blood sample is collected by a blood collection tube as a biological sample used for measurement in a clinical test. The blood collection tube varies depending on the test items, but if serum is required, use a blood collection tube with a coagulation accelerator added.
When plasma is required, a blood collection tube to which heparin is added as an anticoagulant is used. In the case of a blood glucose test, heparin, EDTA (EDTA-2Na, EDTA-2K
), A blood collection tube to which sodium fluoride or the like is added singly or plurally is used, so that the blood sample contains an additive although it is several%. In some inspections, additives often have a problem because they have a great influence on the measurement. In some cases, a blood collection tube may be used for another test, and an EDTA-added blood collection tube used in a blood test may be used for glycated hemoglobin. Therefore, it is desirable to be able to measure regardless of the type of blood collection tube.

[0005]

When a glycated protein in a biological sample is measured by fructosyl amino acid oxidase, a method for measuring a glycated protein using an enzyme which does not inhibit the reaction even if the sample contains EDTA is desired. Was rare. At present, measurement devices in clinical tests have been fully automated, and a measurer has only to set a sample, and it is very easy to obtain a measurement result from sampling a sample. More recently, the measuring apparatus has been connected to a sample transport line to save time and labor. There is a need for a measurement method that is not affected by the blood collection tube used so as to be adaptable to such advanced systems. The method for measuring a saccharified protein in which the saccharified protein is measured by an enzymatic method using fructosyl amino acid oxidase according to the present invention is a method in which a saccharified protein is digested and fragmented into amino acids and peptides with a protease and measured with fructosyl amino acid oxidase. And treating with a protease is known in the field of the present invention.

[0006]

SUMMARY OF THE INVENTION Fructosyl amino acid oxidase which specifically acts on glycated proteins and which does not inhibit the reaction by EDTA within the range of the content normally used in blood collection tubes is used to inhibit bacteria of the genus Fusarium. , Fructosyl lysine or fructosyl N ^α -Z-lysine (hereinafter F
Notated as ZL. ) It could be found by purifying and separating from the supplemented medium. To measure the inhibition of the reaction of fructosyl amino acid oxidase by EDTA, FZL was used as a substrate for fructosyl amino acid oxidase. The reaction is fructosyl amino acid oxidase and EDT
After mixing A in advance, the substrate was added, and the amount of hydrogen peroxide generated by the enzymatic reaction was measured for absorbance by a colorimetric method. The degree of the effect of inhibition was compared using the absorbance value when EDTA was added and the absorbance value without EDTA.

[0007] The medium containing fructosyl lysine and FZL contains 10% glucose and lysine or N ^α -Z-lysine.
It can be obtained by pressing at a high temperature of 0 to 150 ° C. for 3 to 60 minutes. By culturing bacteria using a medium containing fructosyl lysine and FZL, an enzyme having high specificity for fructosyl lysine and FZL can be obtained from the fructosyl amino acid oxidase produced by the bacteria.

When microorganisms in soil were screened using a medium containing fructosyl lysine and FZL, Fusarium oxysporum S-
1F4 ( Fusarium oxysporum S-1F4) (FER
MBP-5010) could be obtained.

The method for producing fructosyl lysine or FZL is described below. Glucose 0.01 to 50% by weight and lysine or N ^alpha -Z- lysine to prepare an aqueous solution containing 0.01 to 20 wt%, to hot pressing treatment 3-60 minutes at a temperature of 100 to 150 ° C. 0.01 ~
0.5% fructosyl lysine or FZL can be obtained. Preferably 200 g glucose, lysine or N
10 g of ^α- Z-lysine as a 1000 ml aqueous solution,
A high temperature and pressure treatment is performed at 120 ° C. for 20 minutes. Subsequently, purification by reverse phase chromatography or ion exchange chromatography can yield fructosyl lysine or FZL. Fructosyl lysine or FZL containing medium
It is obtained by adding purified fructosyl lysine or FZL to the medium.

As a medium to which fructosyl lysine or FZL is added, a normal medium containing a carbon source, a nitrogen source, an inorganic substance and other nutrients can be used. Glucose, xylose, glycerin and the like can be used as the carbon source, and peptone, casein digest, yeast extract and the like can be used as the nitrogen source. As the inorganic substances, those contained in ordinary culture media such as sodium, potassium, calcium, manganese, magnesium, cobalt and the like can be used. The cultivation is performed at 25 to 37 ° C, preferably at 28 ° C. The pH of the medium is in the range of 4.0 to 8.0, preferably 5.5 to 6.0. Fusarium oxysporum S-1F4 under the same conditions
After culturing for 40 hours, preferably for 24 hours, fructosyl amino acid oxidase can be obtained, but culturing for 24 hours is the most efficient culturing time. From the culture thus obtained, it is possible to obtain a purified enzyme product by removing nucleic acids and cell walls. Usually, the enzyme is accumulated in the cells, so the bacteria in the culture are crushed and the enzyme is extracted. The disruption of bacterial cells includes physical means or a dissolving means using a solvent, freezing treatment, ultrasonic treatment, pressurization and the like. The enzyme is separated and purified by appropriately combining salting out with ammonium sulfate or the like, precipitation with an organic solvent such as ethanol, ion exchange chromatography, gel filtration, affinity chromatography and the like. For example, the culture is collected by centrifugation or suction filtration to collect mycelium, washed, suspended in a 0.1 M Tris-HCL buffer (pH 8.5), and crushed with a dynomill. Then, the centrifuged supernatant was fractionated with ammonium sulfate,
Purify by treatment with EAE-Sephacel ion exchange chromatography. The enzyme thus separated from the culture solution and purified is referred to as FAOD-S, and the description will be continued below.

The enzyme produced by Fusarium oxysporum S-1F4 has the following properties: Amadori compound is oxidized in the presence of an enzyme, α-ketoaldehyde,
Catalyze a reaction to produce an amine derivative and hydrogen peroxide;
Stable pH 4.0-12.0, optimal pH 8.0;
Stable temperature is about 20-55 ° C, optimal temperature is 45 ° C;
The molecular weight is about 45000 (45 kDa) as measured by a gel filtration method using a Sephacryl S-200 column.

Fusarium oxysporum S-1F4, a bacterium that produces FAOD-S of the present invention, was isolated from the soil by the present inventors, and has the following mycological properties. For the classification of bacteria, see The Genus
Fusarium (The Genus Fusarium) "(CMI. 1971). Growth in culture medium Growth in PDA medium, potato sucrose agar medium and oatmeal medium was very good in any medium. After culturing at 25 ° C. for 7 days, the cells spread in a felt-like manner over the entire culture vessel and exhibit a white or pale purple color. Taxonomic properties The isolated bacteria were identified by culturing the microorganisms in an oatmeal medium and observing morphology under a microscope such as conidia and (conidiophor). As a result, the color of the colony is white to pale purple, and microcolonia (microc
onidia), macroconidia, and chlamydospores. Macroconidia has a crescent shape, 3 to 5 partitions, and microconidia has an oval or elliptical shape, forming small conidiospores in a pseudohead shape. Therefore, it was identified as Fusarium oxysporum. This bacterium was provided to the National Institute of Bioscience and Biotechnology by the National Institute of Advanced Industrial Science and Technology
Deposited under BP-5010 (Deposit date: February 24, 1994).

The characteristics of FAOD-S produced by Fusarium oxysporum S-1F4 will be described in detail. General Inducing Properties FAOD-S is an inducing enzyme induced by fructosyl lysine or FZL,
Fusarium oxysporum S-1F4 in the presence of ZL
Can be obtained by culturing. FAOD-S
Is unable to induce enzyme by a separately prepared medium glucose and lysine or glucose and N ^alpha -Z- lysine.

Reaction specificity and substrate specificity FAOD-S is represented by R ¹ -CO-CH ₂ + O ₂ + H ₂ O → R ¹ -CO-CHO + R ² -NH + H ₂ O ₂ (where R ¹ is an aldose residue , R ² represents a protein residue). FAOD-S according to the present invention showed the above-mentioned reaction, and particularly showed high specificity for fructosyl lysine or FZL. The purified enzyme was Fusarium oxysporum S-1F
4 was cultured for 24 hours, the culture was filtered by suction, and the mycelium was collected and washed, and then 0.1 M Tris-HCl buffer (pH
8.5) The suspension was suspended in mycelium by Dynomill,
The supernatant was purified by ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography.

PH and temperature conditions Measurement of pH conditions 0.1 M acetic acid, calcium phosphate (KP), Tris-
HCl and glycine-NaOH buffer (pH 4.0-1
2.0), FAOD-S was added, and the mixture was incubated at 30 ° C. for 10 minutes. Then, the activity was measured under normal conditions (30 ° C., pH 8.0). Measurement of Temperature Conditions FAOD-S was added to a temperature condition of 25 to 60 ° C. in a 0.1 M Tris-HCl buffer (pH 8.0), incubated for 10 minutes, and then the activity was measured under normal conditions. As a result of measurement by the method described above, FAOD
The stable pH range of -S is pH 4.0 to 12.0, and preferably 8.0. The stable temperature range is 20 ~
55 ° C. and gradually deactivates above 30 ° C. Further, the enzymatic reaction proceeds efficiently at 30 to 50 ° C, preferably 40 to 50 ° C, more preferably 45 ° C.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Using the enzyme obtained by the above-mentioned method, the degree of the effect of EDTA as a glycolytic inhibitor was confirmed. A solution of EDTA · 2Na was prepared, and hydrogen peroxide generated by fructosyl amino acid oxidase was measured using FZL as a substrate by a quantitative method using peroxidase.

First, the following EDTA and fructosyl amino acid oxidase were added and mixed.
Incubate at 7 ° C for 10 minutes. By adding color solution containing 10mM EDTA · 2Na 100μl 6U / ml FAOD-S 10μl then substrate solution shown below, 10 mM glycated N ^alpha -Z- lysine 100 [mu] l 3 mM 4-aminoantipyrine 30 [mu] l 3 mM N-ethyl -N -(2-Hydroxy-3-sulfopropyl) -3-methylalanine 30 μl 60 U / ml peroxidase 30 μl 0.1 M Tris-HCl buffer (pH 8) 500 μl These were mixed and incubated at 37 ° C. for 30 minutes. The absorbance at 555 nm was measured. E
A control sample to which the same amount of water was added instead of DTA · 2Na was prepared and measured in the same manner to compare the degree of influence of EDTA · 2Na. In addition, measurement was similarly performed for fructosyl amino acid oxidase derived from Gibberella genus known to be inhibited by EDTA · 2Na (hereinafter referred to as FAOD-G).

The absorbance when water was added instead of EDTA was 1.75. Refer to this numerical value for each FA
Looking at the influence of EDTA on OD, FAOD-S
Had no absorbance at 1.78, but F
In AOD-G, the absorbance is 1.05 and the enzyme reaction is EDTA.
Was found to be inhibited by as much as 40%. This proved that FAOD-S was not inhibited by EDTA · 2Na.

[0019]

According to the present invention, by using fructosyl amino acid oxidase derived from Fusarium oxysporum, even if EDTA is contained in the sample, the glycated protein can be accurately detected without inhibiting the enzyme reaction. It became possible to measure.

[0020]

[Brief description of the drawings]

FIG. 1. EDTA of FAOD-S and FAOD-G
The effect of

[Procedure amendment]

[Submission date] July 4, 1997

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All

[Correction method] Added

[Correction contents]

FIG.

Claims (2)

[Claims] (1) When glycated protein in a sample is measured by fructosyl amino acid oxidase,
A method for measuring a glycated protein, wherein the sample is measured using a sample containing EDTA. 2. A bacterium that produces fructosyl amino acid oxidase is Fusarium oxysporum S-1F4 (Fu
sarium oxysporum S-1F4) (FERMBP-50)
The method for measuring a saccharified protein according to claim 1, which is 10).