JP2001204494A - Kit for assaying saccharified hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for assaying saccharified hemoglobin and fructosylvaline which is a degraded product thereof. SOLUTION: This method for assaying fructosylvaline is characterized in that a fructosylvaline oxidase acts on the fructosylvaline under one of the following conditions (a) to (c): (a) the reaction solution having pH <=6; (b) the reaction solution having >=200 mM electrolyte concentration; and (c) the combination of the conditions (a) and (b).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel method for measuring glycated hemoglobin (hemoglobin Alc; HbAlc). More specifically, the present invention relates to a method for measuring HbAlc and its degradation product, fructosyl valine, which is used in the field of clinical testing and the like, and an enzyme sensor system constructed based on the method.

[0002]

2. Description of the Related Art The amino groups of the protein main chain and side chains are non-enzymatically linked to the reducing end of a reducing sugar such as glucose.
Amadori compounds or glycated proteins are produced. It is known that hemoglobin is saccharified in blood to produce glycated hemoglobin (glycated hemoglobin; HbAlc). That the presence of HbAlc in hemoglobin is higher in diabetic patients than in healthy individuals;
Since the blood concentration of bAlc reflects the blood glucose level in the past several weeks, the blood concentration of HbAlc is extremely important in clinical trials as an indicator for diabetes diagnosis and blood sugar control in diabetic patients.

[0003] In HbAlc, fructosyl valine can be used as a low molecular model compound of HbAlc because glucose is bonded to valine at the N-terminal of the hemoglobin β chain. That is, HbAlc can be assayed using an enzyme using fructosyl valine as a substrate.

[0004] Enzymes acting on Amadori compounds have been isolated from various strains, and it has been suggested that glycated proteins such as glycoalbumin, HbAlc and fructosamine can be analyzed using these enzymes. (JP-A-61-268178, JP-A-61-280)
297, JP-A-3-155780, JP-A-5-1921
93, JP-A-7-289253, JP-A-8-15467
2, Agric. Biol. Chem. , 53 (1),
103-110, 1989, Agric. Biol. C
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J. Biol. Chem. , 269 (44), 2729
7-27302, 1994, Appl. Enviro
n. Microbiol. , 61 (12), 4487-
4489, 1995, Biosci. Biotech.
Biochem. , 59 (3), 487-491, 19
95; Biol. Chem. , 270 (1), 21
8-224, 1995; Biol. Chem. , 2
71 (51), 32803-32809, 1996,
J. Biol. Chem. , 272 (6), 3437-
3443, 1997).

However, using the fructosylamine oxidase reported so far, fructosyl valine, which is a fructosylamine compound that is present in glycated hemoglobin and serves as an indicator of glycated hemoglobin content, is used for the production of glycated albumin. It was difficult to distinguish it from fructosyl lysine, a fructosyl amine compound known as a major degradation product.

This is attributable to the substrate specificity of fructosylamine oxidase reported so far. Therefore, it was expected that fructosylamine oxidase could be selectively reacted with fructosyl valine.

[0007]

SUMMARY OF THE INVENTION The present invention provides a novel method for measuring glycated hemoglobin (hemoglobin HAlc; HbAlc) and its degradation product, fructosyl tosyl valine, and an analysis kit and an enzyme sensor system constructed based on the method. I will provide a.

[0008]

Means for Solving the Problems According to the present invention, as a result of intensive studies on the reaction conditions for fructosylamine oxidase, the present inventors have found conditions for selectively reacting fructosyl valine with fructosylamine oxidase.

[0009] That is, for selectively reacting fructosyl valine with fructosyl valine and fructosyl lysine equivalently, or for selectively reacting fructosyl valine with the substrate specificity of fructosylamine oxidase using fructosyl lysine as a substrate. Any of the following reaction conditions a) to c) a) The pH of the reaction solution is 6 or less. b) The electrolyte concentration of the reaction solution is 200 mM or more. c) The reaction conditions by the combination of a) to b) are provided. The present invention also provides a measurement method characterized by measuring fructosyl valine obtained by decomposing HbAlc or fructosyl valine by reacting it with fructosylamine oxidase under the above reaction conditions. .

[0010] The present invention also provides a fructosyl valine assay kit for performing the above-described method.

The present invention also provides a kit for measuring glycated hemoglobin for measuring fructosyl valine by the method described above.

The present invention further provides an enzyme sensor system for measuring HbAlc or fructosyl valine constructed based on the measuring method of the present invention.

The present invention further provides a method for measuring HbAlc or fructosyl valine and an assay kit according to the present invention, wherein the fructosylamine oxidase is Pichia.
sp. Provided is a fructosylamine oxidase derived from N1-1 strain.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The fructosylamine oxidase used in the method of the present invention is not particularly limited as long as the present invention can be achieved. For example, it can be produced using a microorganism that produces fructosylamine oxidase. Examples of such microorganisms include Pichi
a sp. N1-1 strain (FERM P-17326). The microorganism is cultured in a suitable natural or synthetic medium containing fructosyl valine. It is preferable to increase the enzyme yield by inducing fructosyl valine kinase using a medium containing fructosyl valine as the sole nitrogen source. As a medium using fructosyl valine as the sole nitrogen source, a medium obtained by adding fructosyl valine to a minimal medium (for example, M9S) can be mentioned. After recovering the cells from the culture solution by centrifugation or the like, the cells are disrupted by a French press or the like. This is subjected to ultracentrifugation to obtain a water-soluble fraction containing fructosyl phosphatase. The obtained water-soluble fraction is subjected to ion exchange chromatography,
Fructosylamine oxidase is prepared by purification by affinity chromatography, HPLC or the like. The method for measuring fructosyl valine or fructosyl valine produced by enzymatically or chemically decomposing HbAlc according to the present invention is carried out under the following conditions a) to c). Act on these substrates. Fructosylamine oxidase that specifically oxidizes fructosyl valine under the same conditions includes Pichia sp. N1-1
Derived fructosylamine oxidase and the like can be used. a) The pH of the reaction solution is 6 or less. b) The concentration of the electrolyte in the reaction solution is 200 mM or more. c) Reaction conditions by a combination of a) to b).

The substrate selectivity of the fructosylamine enzyme is
When the pH of the reaction solution is around 7, as shown in FIG.
The ratio of the enzyme activity when using fructosyl valine as a substrate to the enzyme activity when using fructosyl lysine as a substrate is 2
The following is a practical problem from the viewpoint of distinguishing these two substrates. However, as the pH decreases, the reaction ratio to fructosyl valine increases, and at pH 6 or less, preferably pH 5.5 or less, more preferably pH 5.0 or less, the specific activity becomes 2 or more, which is sufficient for practical use. Increased selectivity for sylvaline.

Further, the substrate selectivity of fructosylamine oxidase, as shown in FIGS. 2 and 3, when the concentration of the electrolyte in the reaction solution is 200 mM or less, is determined by the ratio of fructosylamine to the enzyme activity when fructosyl lysine is used as a substrate. The ratio of enzyme activity when phosphorus is used as a substrate is 2 or less, and there is a practical problem in discriminating the two substrates. But,
When the electrolyte concentration in the reaction solution increases, the reaction ratio improves, and when the electrolyte concentration of the reaction solution is 200 mM or more, preferably 300 mM or more, more preferably 400 mM or more, the specific activity becomes 2 or more, and it can be sufficiently used for practical use. Increased selectivity for ctucylvaline.

Measurement of enzyme activity Fructosylamine oxidase activity can be measured by quantifying the amount of oxygen consumed by the enzyme reaction or the amount of hydrogen peroxide generated. Various methods for quantifying oxygen and hydrogen peroxide are known in the art. For example, a constant temperature cell is charged with a buffer containing fructosyl valine kinase and a mediator, maintained at a constant temperature, a sample containing fructosyl valine is added thereto, and the color reaction of the redox dye is monitored. And the activity of fructosylamine oxidase can be measured. Alternatively, the generated hydrogen peroxide can be quantified using peroxidase. As such a measurement system, a peroxidase to 4-aminoantipyrine system is known and described in various experiment books (for example, Biological Engineering Experiment Book, edited by Biotechnology Society, Baifukan 1992).

In the same manner, various artificial electron mediators can be used instead of oxygen as the electron acceptor of fructosylamine oxidase. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate, and the like can be used.

Assay Kits In another aspect, the invention features a fructosyl valine assay kit for performing the above methods.
The fructosyl valine assay kit of the present invention contains a reaction solution having the measurement conditions for fructosyl valine kinase according to the present invention in an amount sufficient for at least one assay.
Typically, the kit comprises a fructosylamine oxidase and buffers necessary for the assay according to the conditions of the invention, suitable mediators and, if necessary, enzymes such as peroxidase, for preparing a calibration curve. A standard solution of fructosyl valine or a derivative thereof,
As well as guidelines for use. The fructosyl valine assay kit according to the invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.

In yet another aspect, the invention relates to Hb
Features an Alc assay kit. HbAlc is enzymatically or chemically degraded to produce fructosyl valine, and HbAlc can be assayed by quantifying it using the fructosyl valine analysis kit of the present invention. Therefore, the HbAlc assay kit of the present invention further comprises a hydrolyzing reagent or a protease in addition to the fructosyl valine assay kit described above.

Enzyme sensor system In another aspect, the invention features a sensor system for measuring fructosyl valine and a sensor system for measuring HbAlc. Using the sensor system of the present invention, the concentration of the substrate, fructosyl valine, is measured by measuring the oxygen consumed or the hydrogen peroxide generated by the action of fructosylamine oxidase under the reaction conditions of the present invention. Can be determined. Various sensor systems for measuring oxygen or hydrogen peroxide are known in the art. As an electrode, an oxygen electrode, a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and the enzyme of the present invention is immobilized on the electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of enclosing in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, and a redox polymer. Is also good.

When an oxygen electrode is used, the enzyme is immobilized on the electrode surface, inserted into a buffer, and maintained at a constant temperature. A sample is added thereto and the decrease value of the current is measured.

When the measurement is performed in an amperometric system using a carbon electrode, a gold electrode, a platinum electrode or the like, these electrodes on which an enzyme is immobilized are used as working electrodes, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, a platinum electrode). For example, Ag / Ag
(Cl electrode) together with a buffer containing a mediator to maintain a constant temperature. A constant voltage is applied to the working electrode, a sample is added, and the increase in current is measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate, and the like can be used.

Alternatively, the same enzyme electrode can be used for analysis by measuring hydrogen peroxide generated as a result of the enzyme reaction under the reaction conditions of the present invention. That is, these electrodes on which an enzyme is immobilized are used as working electrodes, and a counter electrode (for example, platinum electrode) and a reference electrode (for example,
g / AgCl electrode) and kept at a constant temperature in a buffer solution under the conditions of the present invention. A constant voltage is applied to the working electrode, the sample is added and the increase in current due to hydrogen peroxide resulting from the enzymatic reaction is measured. FIG. 5 shows a schematic diagram of an enzyme sensor system combining a fructosylamine oxidase and a platinum electrode.

Further, as a method of measuring with an amperometric system using a carbon electrode, a gold electrode, a platinum electrode or the like, there is a system using an immobilized electron mediator. That is, these electrodes in which an enzyme and an electron mediator such as potassium ferricyanide, ferrocene, osmium derivative, and phenazine methosulfate are immobilized on a polymer matrix by adsorption or covalent bonding as a working electrode are used as a counter electrode (for example, a platinum electrode) and a reference electrode. It is inserted into a buffer together with an electrode (eg, an Ag / AgCl electrode) and kept at a constant temperature. A constant voltage is applied to the working electrode, a sample is added, and the increase in current is measured.

Regardless of which electrode is used, the concentration of fructosyl valine in a sample can be determined according to a calibration curve prepared using a standard concentration of a fructosyl valine solution. When used as a sensor system for measuring HbAlc, the above-described sensor system for measuring fructosyl valine is combined with a membrane on which a protease (eg, protease) is immobilized, and the like.
Build a composite sensor system. Such a structure of a composite sensor system using a continuous reaction by a combination of a plurality of enzymes is well known in the art, for example, Biosensors-Fundame.
ntal and Applications-Ant
honey P. F. Tuner, Isao Karub
e and GeorgeS. Wilson, Oxfo
rd University Press 1987.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 minimal medium _{(Na 2 HPO 4 6%,} KH 2 PO 4 0.
3%, NaCl 3%, MgSO ₄ 0.01%, C
aCl ₂ 0.01%, D- glucose 1%) 150m
was cultured in a medium to which fructosyl valine having a final concentration of 0.52% was added as a nitrogen source. Using this medium, P
ichia sp. N1-1 is cultured, and the obtained cells are washed with a 10 mM phosphate buffer solution (pH 7.0). The cells are digested with the enzyme Zymylase to disrupt the cells. The crushed liquid is subjected to ultracentrifugation (4000 r.
pm, 20 min), the supernatant is lyophilized and concentrated. This enzyme concentrate is used as an enzyme sample. Alternatively, an enzyme preparation further purified by anion exchange chromatography can be used. Using the fructosylamine oxidase (FAO) thus obtained,
The pH dependence of the selectivity of fructosyl valine was studied as follows. As a buffer solution, a 0.5 M McIlvaine buffer solution adjusted to pH 4.9 to 8.2 was used. In the activity measurement, hydrogen peroxide generated based on oxidation of fructosylamines was measured using a peroxidase to 4-aminoantipyrine system. FIG. 1 shows the pH dependence of the enzyme activity when fructosyl valine and fructosyl lysine were used as substrates. In this buffer, at a pH of 7.0 or more, the ratio of the enzyme activity to fructosyl valine was 1.5 or less. However, the reaction ratio to fructosyl valine improved with the decrease in pH. At pH 4.9, the ratio of the enzyme activity to fructosyl valine was 5 and the selectivity for fructosyl valine was 5%. Became higher. Example 2 As in Example 1, Pichia sp. F derived from N1-1
The dependence of the selectivity of FAO on the buffer solution concentration was examined using AO. 5m adjusted to pH 7.0 as buffer solution
M-500 mM potassium phosphate buffer solution was used. In the activity measurement, hydrogen peroxide generated based on oxidation of fructosylamines was measured using a peroxidase to 4-aminoantipyrine system. FIG. 2 shows the buffer activity concentration dependence of the enzyme activity when fructosyl valine and fructosyl lysine were used as substrates. 5 mM to 1 in this buffer
At around 00 mM, the ratio of the enzyme activity when using fructosyl valine as a substrate to the enzyme activity when using fructosyl lysine as a substrate was 1.5 or less. However, as the concentration of the buffer solution was increased, the reaction ratio to fructosyl valine was improved. At 500 mM, the ratio of the enzyme activity to fructosyl valine was 8.5, and the ratio of the enzyme activity to fructosyl valine was 8.5. The selectivity was higher. Example 3 As in Example 1, Pichia sp. F derived from N1-1
The dependence of the selectivity of FAO on the NaCl concentration in the buffer solution was examined using AO. As a buffer solution, a 10 mM potassium phosphate buffer solution adjusted to pH 7.0 was used.
The aCl concentration was studied between 0 and 1000 mM. In the activity measurement, hydrogen peroxide generated based on oxidation of fructosylamines was measured using a peroxidase to 4-aminoantipyrine system. Na in buffer solution of enzyme activity using fructosyl valine and fructosyl lysine as substrates
FIG. 3 shows the Cl concentration dependency. N in this buffer
At an aCl concentration of about 0 mM to 50 mM, the ratio of the enzyme activity when using fructosyl valine as a substrate to the enzyme activity when using fructosyl lysine as a substrate was 1.5 or less.
However, as the concentration of NaCl in the buffer solution increased, the reaction ratio to fructosyl valine improved, and at a NaCl concentration of 500 mM, the ratio of the enzyme activity to fructosyl valine was 3%.
And increased selectivity for fructosylvaline. Example 4 Fusallium oxyspor as in Example 2
um f. sp. fragariae (IFO 311
80) The dependence of the selectivity of FAO on the buffer solution concentration was examined using the FAO derived from the method. IFO31180 is a minimal medium (Na ₂ HPO ₄ 6%, KH ₂ PO ₄ 0.3%, N
aCl 3%, MgSO ₄ 0.01%, CaCl ₂
(0.01%, D-glucose 1%) in 150 ml of a medium in which fructosyl lysine having a final concentration of 0.52% was added as a nitrogen source. The obtained cells were washed and crushed with glass beads. This crushed liquid is centrifuged (5000 g,
The supernatant prepared for 20 min) was dialyzed overnight against a 10 mM phosphate buffer solution (pH 7.0), and then concentrated by freeze-drying. The enzyme concentrate thus prepared was used as an enzyme sample. As a buffer solution, a 5 mM to 500 mM potassium phosphate buffer solution adjusted to pH 7.0 was used. In the activity measurement, hydrogen peroxide generated based on oxidation of fructosylamines was measured using a peroxidase to 4-aminoantipyrine system. FIG. 4 shows the buffer activity concentration dependence of the enzyme activity when fructosyl valine and fructosyl lysine were used as substrates. 5m in this buffer
At around M to 50 mM, the ratio of the enzyme activity when using fructosyl valine as a substrate to the enzyme activity when using fructosyl lysine as a substrate was 1.5 or less. However, as the concentration of the buffer solution was increased, the reaction ratio to fructosyl valine was improved. At 500 mM, the ratio of the enzyme activity to fructosyl valine was 8.5, and the ratio of the enzyme activity to fructosyl valine was 8.5. The selectivity was higher. Example 5 As in Example 1, Pichia sp. F derived from N1-1
AO was prepared, and an enzyme sensor system for measuring fructosylamines using the same enzyme was constructed. FIG. 5 shows a configuration diagram of the sensor system. The filter paper was impregnated with 50 μl of a phosphate buffer containing 0.03 U of the same enzyme, and the filter paper was mounted on a 3 mm-diameter platinum electrode manufactured by BAS, which was covered with a dialysis membrane. This enzyme sensor is 500m
M phosphate buffer pH 7.0 or 10 mM phosphate buffer pH 7.0. The measurement was performed by measuring the signal of the enzyme sensor at the time of addition of 1 mM fructosyl valine or fructosyl lysine at 25 ° C. and an applied potential of 600 mV vs.
Hydrogen peroxide generated by oxidation of fructosylamines was measured as Ag / AgCl. The dependence of the selectivity of FAO on the buffer solution concentration was examined. PH as buffer solution
When a 10 m potassium phosphate buffer solution adjusted to 7.0 was used, the ratio of the enzyme activity when using fructosyl valine as a substrate to the enzyme activity when using fructosyl lysine as a substrate was 1.0. However, at 500 mM, the ratio of the enzymatic activity to fructosyl valine was 4.5, and the selectivity to fructosyl valine was increased.

[Brief description of the drawings]

FIG. 1 shows Pichia sp. PH dependence of selectivity of enzymatic reaction to fructosyl valine when using N1-1-derived fructosylamine oxidase

FIG. 2 shows Pichia sp. Dependence of selectivity of enzymatic reaction to fructosyl valine when using N1-1-derived fructosylamine oxidase on phosphate buffer concentration

FIG. 3 shows Pichia sp. Dependence of NaCl concentration on selectivity of enzymatic reaction to fructosyl valine when using N1-1-derived fructosylamine oxidase

FIG. 4 shows Fusallium IFO31 as an enzyme
180 using fructosylamine oxidase
Dependence of selectivity of enzymatic reaction on fructosyl valine on phosphate buffer concentration

FIG. 5 shows Pichia sp. Configuration diagram of enzyme sensor system constructed using fructosylamine oxidase derived from N1-1

Claims (8)

[Claims] 1. A method for measuring fructosyl valine, which comprises reacting fructosyl valine with fructosylamine oxidase under any of the following conditions a) to c): Method for measuring ctucylvaline. a) The pH of the reaction solution is 6 or less. b) The electrolyte concentration of the reaction solution is 200 mM or more. c) Reaction conditions by a combination of a) to b). 2. A glycated hemoglobin (hemoglobin Alc;
A method for measuring HbAlc), comprising quantifying fructosyl valine produced by decomposing HbAlc in a sample by the method according to claim 1. 3. A kit for measuring fructosyl valine for carrying out the method according to claim 1. 4. A kit for measuring glycated hemoglobin for carrying out the method according to claim 1. 5. The method according to claim 1, wherein the electrolyte concentration is 200 mM or more at a pH of 6 or less, and Pichia sp. A fructosyl valine measurement kit containing N1-1-derived fructosylamine oxidase. 6. An enzyme sensor system for fructosyl valine measurement, comprising the method according to claim 1. 7. An enzyme sensor system for measuring glycated hemoglobin, comprising the method according to claim 1. 8. The method according to claim 1, wherein the enzyme is Pichi.
a sp. What is N1-1-derived fructosyl valine kinase.