JP3034984B2 - Highly sensitive method and composition for quantification of D-galactose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive method for quantifying D-galactose, which is useful in clinical biochemical tests, food tests and the like, and a composition for quantifying the same.

[0002]

2. Description of the Related Art The measurement of D-galactose as a clinical test is extremely important for diagnosis of galactosemia (galactosemia), monitoring of a galactose tolerance test during liver function tests, and the like. The measurement methods include chemical methods,
Enzymatic methods and the like are known. As a chemical method, there are a Nylander method and a Benedict method that use the reducing power of galactose. These methods are easy to operate, but have low specificity and a galactose concentration of 50%.
Currently, it is not widely used because there is a problem in sensitivity that it does not give a positive result unless it exceeds mg / dl. On the other hand, as an enzymatic method, galactose dehydrogenase is used.
A method in which the amount of reduced NAD (P) produced is determined by absorbance measurement or fluorescence measurement at 340 nm using coexistence with NAD (P), and the produced hydrogen peroxide is used as a peroxidase and chromogen Colorimetric method in the presence of co-existing with (Japanese clinical practice, 47, 447-449, 1989)
Year special edition).

[0003] All of the above methods are insufficient in sensitivity in view of the normal value of D-galactose.

As a kit for analyzing food, a reagent for measuring D-galactose using galactose dehydrogenase is commercially available from Boehringer Mannheim. Lactose can be measured not only with D-galactose but also with β-galactosidase in food. Is done.

As described above, not only D-galactose in the test solution but also other reaction systems for producing D-galactose can be linked to the enzymatic method of D-galactose. Galactose dehydrogenase is used instead of galactose oxidase.
The reason is that galactose oxidase acts not only on D-galactose but also on D-galactosides. Moreover, galactose dehydrogenase is α, β
Since it acts only on the β-form of the anomeric form, it is used together with mutarotase for the analysis of the anomeric form of β-galactose (Clinical Chemistry, Vol. 10, No. 1, 77-80).
P. 1981).

[0006] On the other hand, β-D-galactosidase is an enzyme widely used as a so-called labeling enzyme in an enzyme immunoassay, and its activity measurement has an important meaning.
Usually, the activity is measured using a chromogenic substrate such as p-nitrophenyl-β-D-galactoside or a fluorescent substance such as 4-methylumbelliferyl-β-D-galactoside. Β-D-galactose, another product of the galactosidase reaction, can also be quantified by using D-galactose dehydrogenase. In fact, an attempt has been made to apply the generated reduced NAD to an ultrasensitive measurement method based on bioluminescence (enzyme immunoassay, p. 66, Medical Shoin, 1987).

In general, when performing analysis using an enzyme, the target substance to be measured is converted into hydrogen peroxide, reduced NAD (P), or the like, which can be detected spectroscopically, as in each of the above methods. In this case, the amount of the detectable substance is stoichiometrically equal to the measurement target.

At present, a method using a spectroscopic analyzer is most widely used as a method for measuring a detectable substance, but this method also has a limit in sensitivity, and is not suitable when the content of the object to be measured is small. There were drawbacks.

Therefore, when the content of the object to be measured is small or when the amount of the specimen containing the object to be measured is small,
Fluorescence analysis, luminescence analysis, and the like, which are more sensitive than spectroscopic analysis, are used. However, these methods are not very suitable for general-purpose examinations such as clinical examinations in terms of the spread of equipment.

As another method for measuring a trace amount of a substance, a so-called enzyme cycling method in which a coenzyme is amplified using two kinds of enzymes when the substance can be converted into an equal amount of a coenzyme or the like. Is being done. For example, the NAD cycling method, CoA cycling method, ATP cycling method, and the like have been used, but none of these methods is practically used in routine analysis such as clinical examinations because the operation is too complicated.

[0011]

If it is possible to improve the sensitivity of the measurement, it is possible not only to reduce the content of the substance to be measured but also to reduce the amount of the sample required for the measurement. In the case where a substance containing various components is used for a subject, the influence of a coexisting substance on the measurement system can be reduced. In addition, it is possible to increase the number of items that can be tested with a limited sample amount, and furthermore, when the sample is human blood, it is possible to reduce the amount of blood collected, so that the Burden can be reduced. In this way, increasing the detection sensitivity is considered from the necessity of using a valuable sample called blood and the need to measure trace components in clinical tests.
It is an inevitable request.

As described above, the conventional method for quantifying D-galactose is not yet satisfactory, and development of a highly sensitive and simple method for quantification has been desired.

Accordingly, a first object of the present invention is to provide a highly sensitive and simple method for quantifying D-galactose.

Further, a second object of the present invention is to provide the above-mentioned D-
An object of the present invention is to provide a composition which is suitably used for a highly sensitive method for quantifying galactose.

[0015]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, in the quantification of D-galactose, a group consisting of thio-NADs and thio-NADPs (hereinafter referred to as thio-type NAD (P ))
One group consisting of NADs and NADPs (hereinafter referred to as non-thio N
AD (P) may be used) galactose dehydrogenase acting on one coenzyme selected from the group consisting of thio-type NAD (P) and non-thio-type NAD (P) By, it can be found that good enzyme cycling can be achieved, and at the time of absorbance measurement, reduced thio NAD (P)
And the reduced non-thio NAD (P) each have an absorption wavelength of 4
Since it is different from around 00 nm and around 340 nm, it can be measured based on any of these absorption wavelengths, and it is possible to carry out an enzyme cycling reaction that can avoid congestion of the absorption wavelength of other substances, and as a result, high sensitivity measurement is possible. The inventors have found that the present invention has been completed.

That is, the present invention relates to a method for preparing a subject containing D-galactose, wherein one of the following (1) to (3): (1) one selected from the group consisting of thioNADPs and thioNADs; Contains one selected from the group consisting of NADs as a coenzyme, and contains D-galactodehydrogenase (2) A ₁ (3) B ₁ which forms a reversible reaction to generate D-galactonolactone using at least D-galactose as a substrate And reacting it with the following reaction formula [1]

[0017]

Embedded image

(Wherein A ₁ represents thioNADPs, thioNADs, N
Shows the ADP compound or an NAD, A ₂ is a reduced product of A _1, B ₁ is a reduced NADP or reduced NAD when A ₁ is a thio-NADP or thio-NAD, and A ₁ is NADP
When the kind or NAD compound indicates reduced thio-NADP or reduced thio-NAD, B ₂ denotes the oxidized product of B ₁₎
The present invention provides a method for quantifying D-galactose, which comprises forming a cycling reaction represented by the following formula, and measuring the amount of A ₂ or B ₁ changed by the reaction.

Further, the present invention provides a composition for quantifying D-galactose, which comprises the above (1), (2) and (3).

The galactose dehydrogenase (EC 1.1.1.48, 1.1.1.120) used in the present invention has at least D
-Galactose + NAD (P) ⁺ = D-galactonolactone +
NAD (P) H + H ⁺
Selected from the group consisting of NADs and thio NADs, and NAD
Any one can be used as long as it is a coenzyme with one selected from the group consisting of Ps and NADs.

The enzyme of EC 1.1.1.48 is an enzyme specific to (thio) NADs, and is an enzyme specific for Pseudomonas fluorescens (Pse
udomonasfluorescens), Pseudomonas saccharophila, and the like, but enzymes derived from Pseudomonas fluorescens have 24% of NAD activity against NADP (Clinical Enzyme Handbook, p. 198, Kodansha, 1982) ).

The enzyme of EC 1.1.1.120 is (thio) NA
Pseudomonas saccharophila (Pseud
omonas saccharophila), Pseudomonas putida (Pse
udomonas putida).

Among these enzymes, the activity of the enzyme derived from Pseudomonas fluorescens, which is commercially available from Boehringer Mannheim, with respect to the coenzyme is 40% with Tris-HCl (pH 8.0) assuming 100% when NAD is used. 69% for thio NAD, 2% for acetyl NAD
9%, about 53% for deamino NAD, and about 33% for thio NADP.

In the present invention, enzymes of other origins other than the above-mentioned enzymes can be appropriately used as long as they have reactivity with D-galactose as a substrate. Can be appropriately confirmed using a coenzyme and a substrate.

The reaction product of the above enzyme is D-galactono-1,5-lactone, which changes to D-galactono-1,4-lactone depending on pH conditions, or non-enzymatically converts to D-galactonic acid. Hydrolyzed (Methods in Enzymol
ogy, 89, 176). Thus, the reaction product can take various forms, but the enzyme cycling reaction can proceed under the conditions described below, and as a result, highly sensitive quantification of D-galactose has become possible.

In the present invention, the coenzymes represented by A ₁ and B ₂ are thioNADPs, thioNADs, NADPs, and NADs.
ThioNADPs or thioNADs, for example, thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide hypoxanthine dinucleotide phosphate; and thionicotinamide adenine dinucleotide (thioNAD) Thionicotinamide hypoxanthine dinucleotide. Examples of NADPs or NADs include nicotinamide adenine dinucleotide phosphate (NA
DP), acetylpyridine adenine dinucleotide phosphate (acetyl NADP), acetylpyridine hypoxanthine dinucleotide phosphate, nicotinamide hypoxanthine dinucleotide phosphate (deamino NAD)
P); and nicotinamide adenine dinucleotide (N
AD), acetylpyridine adenine dinucleotide (acetyl NAD), acetylpyridine hypoxanthine dinucleotide, and nicotinamide hypoxanthine dinucleotide (deamino NAD). The reduced forms of these coenzymes are thioNADPHs, thioNADHs, NADPHs, NA
Display as DHs.

In the present invention, for A ₁ and B ₁ , for example, when A ₁ is a thioNAD (P), B ₁ is NAD
(P) that is necessary is H class, use one thio-type coenzyme in relation of A ₁ and B _1.

When the galactose dehydrogenase used for quantification uses only (thio) NADs as coenzymes,
When the galactose dehydrogenase used is a coenzyme using only (thio) NADPs from the above-mentioned thio NADs and NADs, the galactose dehydrogenase to be further used is (thio) NADs than the above-mentioned thio NADPs and NADPs. When both co-enzymes and (thio) NADPs are used as coenzymes, the above-mentioned thio-NADs and thio-NADPs and the above-mentioned NADs and NADPs may be appropriately selected, and their oxidized and reduced forms may be appropriately used.

Further, by using the quantification method of the present invention, it is also possible to measure the substrate and the enzyme activity in an enzyme system which releases and produces D-galactose and D-galactose originally contained in the test solution. . Furthermore, the substrate and its enzymatic activity in an enzyme system that can be linked to an enzyme system that releases and produces D-galactose can be also measured. These enzyme systems are not particularly limited, and include, for example, various reaction systems shown below.

(1) An enzyme reaction system between β-D-gaclatoside and β-D-gaclatosidase (EC 3.2.1.23). In this system, the amount of β-D-galactoside or the amount of β-D-galactoside is determined by quantifying the amount of β-D-galactose released or generated.
The activity of D-galactosidase can be measured.
β-D-galactoside + H ₂ O → β-D-galactose +
ROH (R: aglycone)

(2) An enzyme reaction system between α-D-galactose and mutarotase (EC 5.1.3.3). In this system, the amount of α-D-galactose or the activity of mutarotase can be measured by quantifying the amount of β-D-galactose released and produced. α-D-galactose →
β-D-galactose

(3) In the enzyme reaction system of the above (2),
When α-D-galactose is derived from an enzyme reaction system between α-D-galactose-1-phosphate and alkaline phosphatase (EC 3.1.3.1). In this system, by finally quantifying β-D-galactose, α-D
-Galactose-1-phosphate can be quantified or alkaline phosphatase activity can be measured. α-D-
Galactose-1-phosphate + H ₂ O → α-D-galactose + phosphate

In the quantitative composition of the present invention, A ₁
And the concentration of B ₁ represents 0.02～100MM, especially 0.05~20mM
And the amount of galactose dehydrogenase is preferably 1 to 1000 u / ml, particularly preferably 2 to 400 u / ml.
The amount can be appropriately determined depending on the type of the subject and the like, and a larger amount can be used.

In the present invention, the amounts of A ₁ and B ₁ are excessive compared to the amount of D-galactose in the subject, and the amounts of A ₁ and B _{1 of} galactose dehydrogenase
₁ is required to be in excess amount compared to the Km value for each, in particular 20 to 10,000 moles of the test component.

In addition, the quantification method of the present invention is applied to a case where galactose dehydrogenase uses both (thio) NADs and (thio) NADPs as coenzymes.
Combination of NADs and NADs or NADPs, or thioNA
When you select a combination of DP compound and an NAD or NADP compound is further not act on D- galactose to the subject, and using a second dehydrogenase to form a reaction of B ₂ → B _1, further said by exerting a substrate of second dehydrogenases may allowed form cycling reaction by allowed to impart reaction system for reproduction B ₁ between B ₁ and B _2.

That is, a D-galactose-containing specimen is composed of one of the following (1) to (4) (1) one selected from the group consisting of thioNADPs and thioNADs, and one consisting of NADPs and NADs. A galactose dehydrogenase that forms a reversible reaction to generate D-galactonolactone using at least D-galactose as a substrate with one selected from the group as a coenzyme (2) A ₁ (3) B ₁ and / or B ₂ (4) A second dehydrogenase which does not act on D-galactose and forms a reaction of B ₂ → B ₁ and a reagent containing a substrate of the second dehydrogenase are allowed to act, and the following reaction formula is obtained.

[0037]

Embedded image

(Wherein A ₁ represents thioNADPs, thioNADs, N
Shows the ADP compound or an NAD, A ₂ is a reduced product of A _1, B ₁ is a reduced NADP or reduced NAD in the case of A ₁ is a thio-NADP or thio-NAD, and A ₁ is NADP
When the kind or NAD compound indicates reduced thio-NADP or reduced thio-NAD, B ₂ represents an oxidized product of B _1,
B ₂ → B ₁ indicates an enzymatic reaction that produces B ₁ using B ₂ as a coenzyme) to determine D-galactose by forming a cycling reaction represented by the following formula:

In this case, the second dehydrogenase does not substantially act on A ₁ in this assay system, or is used under conditions that do not substantially act on A _1. select is preferred, for example, a combination of selecting the second dehydrogenase does not use the a ₁ essentially as a coenzyme, conditions in which the second dehydrogenase by quantitative relation of a ₁ and B ₂ does not act on the substantially a ₁ And the like. At the time of quantification, the amount of A ₂ generated by the reaction is measured.

[0040] In the above quantitative composition using component (4), the concentration of A ₁ is 0.02～100MM, especially 0.05 to
20mM is preferred, the concentration of B ₂ or / and B ₁ is zero.
The concentration of galactose dehydrogenase is preferably 1 to 1000 u / ml, particularly preferably ₂ to 400 u / ml, and the second dehydrogenase is 20 times the Km value (in mM) for B2 (in units of mM). u / ml unit) or more.
It is preferably 0 u / ml, and the substrate for the second dehydrogenase is preferably in excess, for example, 0.05 to 20 mM. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

The second dehydrogenase is added to supplement the regeneration of B ₁ , which makes it possible to reduce the amount of B ₁ used, and is particularly effective when B ₁ is expensive. . Further, a mixture of B ₂ or B ₁ and B ₂ the reaction may be carried out using instead of B _1. In this case, the amount of B ₁ and / or B ₂ is not particularly limited, but is generally 1/10 mol or less, preferably 1/100 mol or less of A ₁ .

As the second dehydrogenase and its substrate, for example, when B ₂ is a NAD or a thio NAD,
Alcohol dehydrogenase (EC 1.1.1.1) and ethanol, glycerol dehydrogenase (EC 1.1.1.6) (E.Col.
i) and glycerol, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) (from rabbit muscle) and L-
Glycerol-3-phosphate, malate dehydrogenase
(EC 1.1.1.37) (derived from porcine and siu myocardium) and L-malic acid, glyceraldehyde phosphate dehydrogenase (EC
1.1.1.12) (rabbit skeletal muscle, liver, yeast, when E.Coli derived) and D- glyceraldehyde phosphate and phosphoric acid, B ₂ is a NADP or the thio-NADP, glucose-6-phosphate dehydrogenase ( EC 1.1.1.49) (derived from yeast) and glucose-6-phosphate, isocitrate dehydrogenase (EC 1.
1.1.42) (derived from yeast and porcine myocardium) and isocitrate and glyoxylate dehydrogenase (EC 1.2.1.17) (Pseudomona
soxalaticus), CoA and glyoxylic acid, phosphogluconate dehydrogenase (EC 1.1.1.44) (rat liver,
Brewer's yeast, from E. Coli) and 6-phospho-D-gluconic acid, glyceraldehyde phosphate dehydrogenase (EC 1.
2.1.13) (derived from plant chloroplasts), D-glyceroaldehyde-3-phosphate and phosphoric acid.

The method of the present invention is characterized in that galactose dehydrogenase is used for (thio) NADs and (thio) NADP.
When both coenzymes are coenzymes, the combination of thioNADs and NADs or NADPs,
When a combination of NADPs and NADs or NADPs was selected, D-galactose did not act on the subject, and A
₂ → Use a third dehydrogenase that forms a reaction of A ₁ , and further provide a reaction system for the regeneration of A ₁ between A ₁ and A ₂ by allowing a substrate of the third dehydrogenase to act. This can form a cycling reaction.

That is, D-galactose-containing specimens include (1) one selected from the group consisting of thioNADPs and thioNADs, and one selected from NADPs and NADs. A galactose dehydrogenase that forms a reversible reaction to produce D-galactonolactone using at least D-galactose as a substrate, and (2) A ₁ or / and A ₂ (3) B ₁ (5 ) A third dehydrogenase which does not act on D-galactose and forms a reaction of A ₂ → A ₁ and a reagent containing a substrate of the third dehydrogenase are allowed to act on the reaction.

[0045]

Embedded image

(Wherein A ₁ represents thioNADPs, thioNADs, N
Shows the ADP compound or an NAD, A ₂ is a reduced product of A _1, B ₁ is a reduced NADP or reduced NAD in the case of A ₁ is a thio-NADP or thio-NAD, and A ₁ is NADP
When the kind or NAD compound indicates reduced thio-NADP or reduced thio-NAD, B ₂ represents an oxidized product of B _1,
A ₂ → A ₁ indicates an enzymatic reaction that generates A ₁ using A ₂ as a coenzyme) to form a cycling reaction represented by the following formula:

In this case, the third dehydrogenase is used in such a manner that it does not substantially act on B ₁ in this assay system, or that it does not substantially act on B _1. the combination is preferably, for example, a combination of selecting an enzyme which does not use B ₁ essentially as a coenzyme, to the quantitative relationship between B ₁ and a ₂ third dehydrogenase which select a condition that does not act to substantially B ₁ Etc. are exemplified. When quantitative measures the consumption of B _1.

[0048] In the quantitative composition using the component (5), the concentration of B ₁ represents 0.02～100MM, especially 0.05 to
20mM is preferred, the concentration of A ₂ or / and A ₁ is 0.
The concentration of galactose dehydrogenase is preferably 1 to 1000 u / ml, particularly preferably ₂ to 400 u / ml, and the third dehydrogenase is 20 times the Km value (mM unit) for A2. (U / ml unit) or more.
0 u / ml is preferred, and the substrate for the third dehydrogenase is preferably in excess, for example, 0.05 to 20 mM. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

The third dehydrogenase is supplementarily added for the regeneration of A ₁ , which makes it possible to reduce the amount of A ₁ used, and is particularly effective when A ₁ is expensive. . Further, the reaction may be carried out using a mixture of A ₂ or A ₁ and A ₂ instead of A _1. In this case, the use amount of A ₁ and / or A ₂ is not particularly limited, but is generally 1/10 mol or less, preferably 1/100 mol or less of B ₁ .

As the third dehydrogenase and its substrate, for example, when A ₁ is a NAD or a thio NAD, alcohol dehydrogenase (EC 1.1.1.1) and acetaldehyde, glycerol dehydrogenase (EC 1.1.1.
6) (from E. Coli) and dihydroxyacetone, L-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) (from rabbit muscle) and dihydroxyacetone phosphate, malate dehydrogenase (EC1.1.1.37) (pig) Heart muscle, bovine heart muscle) and oxaloacetate, glyceroaldehyde phosphate dehydrogenase (EC 1.1.1.12) (from sagittal skeletal muscle, liver, yeast, E. coli) and 1,3-diphospho-D-glyceric acid, A ₁ In the case of NADPs or thioNADPs, glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) (derived from plant chloroplasts), 1,3-diphospho-D-glyceric acid and the like can be mentioned.

Regarding the composition of the reaction solution, two kinds of coenzymes are appropriately selected in consideration of the relative activities between various coenzymes of the galactose dehydrogenase to be used, and then the optimum pH conditions for the forward reaction / reverse reaction are determined by enzyme cycling. What is necessary is just to set so that a reaction may progress efficiently. The enzymes used here may be used alone or in combination of two or more as appropriate.

In order to measure D-galactose in a subject using the composition for quantification thus prepared according to the present invention, the components (1) to (3), (1) to (4), or ( 0.001 to 0.5 ml of an analyte is added to the composition containing 1) to (3) and (5), and the mixture is reacted at a temperature of about 37 ° C. 1 minute after 3 minutes and 4 minutes, or 3 minutes and 8 minutes
The amount or consumption amount of B ₁ of A ₂ generated in 5 minutes after minute, may be measured by the change in absorbance based on the respective absorption wavelengths. For example, A ₂ is a thio NAD
When H and B ₁ are NADH, the production of A ₂ is measured by increasing the absorbance around 400 nm, or the consumption of B ₁ is 3
Measured by the decrease in absorbance around 40 nm, a known concentration of D
-The respective amounts in the sample liquid can be determined in real time by comparing with the values measured using galactose.

Further, the quantification method of the present invention can be applied to a method for measuring D
-The galactose itself leads to the enzyme cycling reaction, and is less susceptible to coexisting substances in the test solution, so that a blank measurement of the test solution can be omitted;
A simple measurement by the late assay can be performed.

In the present invention, in the measurement of A ₂ or B ₁ , quantification can be performed by using other known measuring methods instead of the absorbance measurement.

[0055]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto.

Example 1 Determination of D-galactose <Reagent (1)> 100 mM phosphate buffer (pH 6.5) 0.2 mM thioNAD (manufactured by Sigma) 50 u / ml D-
Galactose dehydrogenase (Boehringer Mannheim; Pseudomonas fluorescens (P
seudomonas fluorescens) <Reagent (2)> 10 mM Tris-HCl buffer (pH 8.5) 20 mM reduced NAD (manufactured by Oriental Yeast) <Operation> Put 0.9 ml of reagent (1) in a cuvette, 2
20 μl of 0, 30, 40, and 50 μM D-galactose solutions (manufactured by Tokyo Kasei) were added, and the mixture was added at 37 ° C. for 10 minutes.
Allowed to react for minutes. Thereafter, 0.1 ml of the reaction solution (2) was added to start the enzyme cycling reaction. The absorbance at 400 nm at the second and fifth minutes after the addition of the reagent (2) was read, and the difference was determined. When the concentration of 0 was used as a reagent blank, the value of the reagent blank was subtracted from each result, and the results are shown in FIG. As is clear from FIG.
The change in absorbance with respect to the amount of galactose showed good linearity.

Example 2 Determination of D-galactose <Reagent (1)> 100 mM phosphate buffer (pH 6.5) 0.4 mM thioNADP (manufactured by Sigma) 2 mM EDTA 50 u / ml D-galactose dehydrogenase (Boehringer) Mannheim; Pseudomonas fluorescens (P
seudomonas fluorescens) <Reagent (2)> 10 mM Tris-HCl buffer (pH 8.5) 20 mM reduced NAD (manufactured by Oriental Yeast) <Operation> 0.9 ml of reagent (1) is placed in a cuvette, and 0, 1
20 μl each of 0, 20, 30, 40, and 50 μM D-galactose solutions (manufactured by Tokyo Chemical Industry) were added, and reacted at 37 ° C. for 7 minutes. Thereafter, 0.1 ml of the reaction solution (2) was added to start the enzyme cycling reaction. The absorbance at 400 nm at the second and fifth minutes after the addition of the reagent (2) was read, and the difference was determined. When the concentration of 0 was used as a reagent blank, the value of the reagent blank was subtracted from each result,
The result is shown in FIG. As is apparent from FIG.
-The amount of change in absorbance with respect to the amount of galactose showed good linearity.

Example 3 Determination of D-galactose <Reagent> 100 mM phosphate buffer (pH 6.5) 0.2 mM thioNAD (Sigma) 4 mM reduced NADP (Oriental Yeast) 2 mM EDTA 35 u / Ml D-galactose dehydrogenase (Boehringer Mannheim; Pseudomonas fluorescens (P
<Operation> Take 1 ml of the reagent in a cuvette, and place 0, 20, 40, 60,
20 μl of 80 and 100 μM D-galactose solutions (manufactured by Tokyo Kasei) were added, and the reaction was started at 37 ° C. The absorbance at 400 nm at the second and fifth minutes after the start of the reaction was read to determine the difference. When the concentration of 0 was used as a reagent blank, the value of the reagent blank was subtracted from each result, and the results are shown in FIG. As is clear from FIG. 3, the amount of change in absorbance with respect to the amount of D-galactose showed good linearity.

[0059]

As described above, since the present invention uses coenzymes having different reduced absorption wavelengths, it is possible to significantly increase the measurement sensitivity without causing measurement errors and by combining enzyme cycling reactions. It becomes possible. As a result, D-galactose in the subject can be simply and accurately determined using only a small amount of the sample.

[Brief description of the drawings]

FIG. 1 is a drawing showing the results of a late assay at 400 nm with respect to the amount of D-galactose in Example 1.

FIG. 2 is a drawing showing the results of a late assay at 400 nm with respect to the amount of D-galactose in Example 2.

FIG. 3 is a drawing showing the results of a late assay at 400 nm with respect to the amount of D-galactose in Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/25-1/66 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A test sample containing D-galactose,
The following (1) to (3) (1) one selected from the group consisting of thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thioNADPs) and thionicotinamide adenine dinucleotides (hereinafter referred to as thioNADs) , One selected from the group consisting of nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADPs) and nicotinamide adenine dinucleotides (hereinafter referred to as NADs) as a coenzyme and at least D-galactose as a substrate using D-galactose as a substrate. Galactose dehydrogenase which forms a reversible reaction to produce nolactone (2) A ₁ (3) A reagent containing B ₁ is allowed to act, and the following reaction formula [1] is used. (Wherein A ₁ represents thioNADPs, thioNADs, NADPs or N
Shows the AD compound, A ₂ is a reduced product of A _1, reduced form NADP When the B ₁ represents A ₁ is thio-NADP or thio-NAD
The kind or a reduced NAD, also when A ₁ is a NADP or NAD compound indicates reduced thio-NADP or reduced thio-NAD, represented by B ₂ denotes the oxidized product of B ₁₎ A method for quantifying D-galactose, which comprises forming a cycling reaction and measuring the amount of A ₂ or B ₁ changed by the reaction. 2. The method of claim 2, wherein the NADPs are nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetyl NADP),
The method for quantifying D-galactose according to claim 1, which is selected from the group consisting of acetylpyridine hypoxanthine nucleotide phosphate and nicotinamide hypoxanthine dinucleotide phosphate (deamino NADP). 3. The NAD is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl NAD), acetylpyridine hypoxanthine dinucleotide and nicotinamide hypoxanthine dinucleotide (deamino NAD). The method for quantifying D-galactose according to claim 1, wherein 4. The method for quantifying D-galactose according to claim 1, wherein the thio NADPs are selected from the group consisting of thionicotinamide adenine dinucleotide phosphate (thio NADP) and thionicotinamide hypoxanthine dinucleotide phosphate. . 5. The method for quantifying D-galactose according to claim 1, wherein the thioNADs are selected from the group consisting of thionicotinamide adenine dinucleotide (thioNAD) and thionicotinamide hypoxanthine dinucleotide. 6. The following components (1) to (3) (1) a coenzyme consisting of one selected from the group consisting of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs. And a galactose dehydrogenase that forms a reversible reaction for producing D-galactonolactone using at least D-galactose as a substrate. (2) A ₁ (3) A composition for quantifying D-galactose, which comprises B ₁ .