Method and reagent for determination of dehydrogenase or its substrate

A method for determining dehydrogenase or its substrate in dehydrogenase reaction, which comprises subjecting a dehydrogenase and its substrate to dehydrogenase reaction in the presence of an electron carrier or a combination of a coenzyme and an electron carrier, allowing to form H.sub.2 O.sub.2 through transfer of electrons derived from the substrate by the dehydrogenase reaction to O.sub.2 via the electron carrier or via the coenzyme and electron carrier, stopping the dehydrogenase reaction, reacting said H.sub.2 O.sub.2 with a peroxidase and a chromogen, and measuring a formed dye, and a reagent for determining dehydrogenase or its substrate. By the method and the reagent, dehydrogenase or its substrate can be accurately determined in a short time regardless of the concentration of the dehydrogenase and/or the substrate and without staining tubes or cells of a determination device with a dye.

The present invention relates to a method for determining dehydrogenase or 
its substrate in the dehydrogenase reaction wherein the dehydrogenase or 
the substrate can be determined accurately and quickly regardless of its 
concentration without staining the determination device with dye or the 
like, and a reagent for use in the method for the determination of the 
dehydrogenase or its substrate. 
PRIOR ART 
There have been known various dehydrogenation reactions catalyzed by 
various dehydrogenases. For example, the reaction of converting 
glucose-6-phosphate into glucono-.delta.-lactone-6-phosphate and the 
reaction of converting lactic acid into pyruvic acid are catalyzed by 
glucose-6-phosphate dehydrogenase and lactate dehydrogenase, respectively. 
In these reactions, the determination of the enzyme or the substrate has 
been carried out by, for example, determining reduced coenzymes (e.g. 
NADH, NADPH and the like) which are generated by the reduction of 
coenzymes participating in these reactions (e.g. NAD.sup.+, NADP.sup.+ and 
the like). The determination of the enzyme or the substrate is carried out 
by measuring an absorbance of, for example, NADH. However, these methods 
have a disadvantage that the sensitivity is not sufficient. For this 
reason, there has been introduced a method where the enzyme or the 
substrate is determined in such a way that the above NADH is further 
converted into another compound to be measured. One of these methods is 
the following method utilizing formazan development: 
##STR1## 
wherein 1-mPMS means 1-methoxy-5-methylphenazinium methylsulfate, 
1-mPMSH.sub.2 is a reduced type thereof, MTT means 
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and 
MTTH.sub.2 is a reduced type thereof (formazan dye). 
In the above reaction, NADH generated in the dehydrogenase reaction of 
D-glucose-6-phosphate to D-glucono-.delta.-lactone-6-phosphate is 
converted into NAD.sup.+ by donating electrons to the electron carrier 
such as 1-mPMS (NADPH also reacts in the same manner). 1-mPMS is then 
reduced to 1-mPMSH.sub.2. Subsequently, MTT present in the reaction system 
is reduced by the 1-mPMSH.sub.2 to MTTH.sub.2 or the formazan dye, purple 
coloring of which is measured with a spectrophotometer to determine the 
NADH, and thus the enzyme or the substrate is indirectly determined. In 
addition to the above 1-mPMS, the electron carrier also includes phenazine 
methosulfate (PMS), 9-dimethylaminobenzo-.alpha.-phenazoxonium (Meldola 
blue) and the like. The method by the measurement of formazan dye indeed 
has a higher sensitivity than that of the above method of the 
determination of NADH, but it also has a disadvantage that it is difficult 
to determine with an automatic analyzer since this dye strongly sticks to 
tubes or cells of the determination device 
In order to avoid the above stain with the dye, a method is disclosed in 
Japanese Patent First Publication (KOKAI) No. 83598/1985. According to the 
method, oxygen, peroxidase (POD) and chromogen [e.g. 
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS) and 
4-aminoantipyrine (4-AA)] are present in the reaction system in place of 
MTT in the method by the measurement of formazan development. The former 
part of the reaction is the same as in the method by the determination of 
formazan development. The reaction after the formation of NADH is 
illustrated by the following reaction scheme wherein the electron carrier 
is 1-mPMS and the chromogen is a combination of TOOS and 4-AA. 
##STR2## 
Also in the above reaction system, the electrons are transferred from NADH 
to 1-mPMS to produce 1-mPMSH.sub.2, which then transfers the electrons to 
O.sub.2 present in the reaction system to form H.sub.2 O.sub.2 via 
O.sub.2.sup.-. H.sub.2 O.sub.2 acts on TOOS and 4-AA in the presence of 
peroxidase and thereby the chromogens are oxidized to produce a dye. 
Although this dye can be determined without stain of the determination 
device unlike the formazan dye, this method still has a disadvantage to be 
overcome. That is, the present inventors have tested the above method and 
have found that it has been difficult to obtain an accurate determination 
due to drastic color depression after the color development, especially in 
case of a high concentration of the substrate. 
BRIEF SUMMARY OF THE INVENTION 
An object of the present invention is to take away the above disadvantages 
and to provide a method by which the enzyme or its substrate in the 
dehydrogenase reaction can easily be determined in an accurate and simple 
manner without staining the determination device with the dye and the 
like. Another object of the present invention is to provide a method for 
the determination of the enzyme or its substrate in the dehydrogenase 
reaction regardless of their concentration in the test sample A further 
object of the present invention is to provide a reagent for use in the 
method for the determination of dehydrogenase or its substrate of the 
present invention. These and other objects and advantages of the present 
invention will be apparent to those skilled in the art from the following 
description.

DETAILED DESCRIPTION OF THE INVENTION 
Before explaining the present invention, there are shown experimental 
results of the method described in the above Japanese Patent First 
Publication No. 83598/1985 which were carried out by the present inventors 
as follows: 
[Experiment]Determination of glucose-6-phosphate 
______________________________________ 
Reaction components (1): 
Tris-hydrochloric acid buffer (pH 8.0) 
1.4 ml 
Glucose-6-phosphate solution (0-0.01 M) 
0.1 ml 
Glucose-6-phosphate dehydrogenase solution 
0.3 ml 
(0.5 U/ml) 
NAD.sup.+ solution (0.1 M) 
0.2 ml 
1-mPMS solution (0.5 mg/ml) 
0.1 ml 
Reaction components (2): 
4-Aminoantipyrine solution (10 mg/ml) 
0.3 ml 
TOOS solution (10 mg/ml) 0.3 ml 
Peroxidase solution (100 U/ml) 
0.3 ml 
______________________________________ 
The above reaction components (1) were mixed and reacted at 37.degree. C. 
for just 5 minutes and thereto the reaction components (2) were added and 
the mixture was reacted for further 4 minutes. The absorbance of this 
reaction mixture was measured at 550 nm. FIG. 1 shows a relationship 
between the substrate (glucose-6-phosphate) concentration and OD value 
(value obtained by deducting a value in blank from the found value; shown 
as .DELTA.OD.sub.550). 
As is clear from FIG. 1, a linear relationship between the substrate 
(glucose-6-phosphate) concentration and the OD value is observed at a 
relatively low concentration (below 0.0003 M) of the substrate, but in 
case of a high concentration (0.005 M and 0.010 M) of the substrate, an 
increase of the absorbance was not observed after 4 minutes as in the case 
of the concentration of 0 M due to drastic color depression after color 
development. 
As a result of the present inventors' study, it is assumed that the drastic 
color depression of the developed dye in case of the high substrate 
concentration is due to the side reaction as shown in the following 
diagram 1, and further that in case of another type of reaction, i.e. a 
reaction of sarcosine dehydrogenase, where no coenzyme participates in the 
reaction, a reaction as shown in the following diagram 2 proceeds and 
hence the color depression occurs like that in the above 
glucose-6-phosphate dehydrogenase reaction. 
##STR3## 
In the above diagram 1, electrons generated in the dehydrogenase reaction 
are transferred to the coenzyme and the electron transfer system, and then 
to O.sub.2. However, when the substrate concentration is so high that the 
enzyme reaction is not stopped, (1) the electrons from the reduced 
electron carrier are transferred to the dye (formed by the reaction with 
peroxidase) not to O.sub.2, which results in the drastic color depression, 
and (2) the electrons from the reduced electron carrier are not 
transferred to O.sub.2 but to TOOS, and thereby TOOS is reduced and 
becomes incapable of color development, which results in poor color 
development. The above is the cause of the poor color development and the 
drastic color depressioh in the reaction system. The same principle as 
above will be applicable to the case of diagram 2. After taking these 
matters into consideration, the present inventors have found that the 
electrons from the electron transfer system could be transferred to 
O.sub.2 completely (a) by setting the reaction system so that the 
dehydrogenase reaction and the formation of H.sub.2 O.sub.2 are completed 
before the peroxidase reaction starts; and (b) by adding the chromogen 
after the dehydrogenase reaction is stopped. 
Thus, the present invention relates to a method for determining a 
dehydrogenase or its substrate in a dehydrogenase reaction, which 
comprises subjecting a dehydrogenase and its substrate to dehydrogenase 
reaction in the presence of an electron carrier or a combination of a 
coenzyme and an electron carrier, allowing to form H.sub.2 O.sub.2 through 
transfer of electrons derived from the substrate by the dehydrogenase 
reaction to O.sub.2 via the electron carrier or via the coenzyme and 
electron carrier, stopping the dehydrogenase reaction, reacting said 
H.sub.2 O.sub.2 with a peroxidase and a chromogen, and measuring a formed 
dye. The present invention relates further to a reagent for use in the 
determination of substrate of dehydrogenase, comprising (i) a 
dehydrogenase, (ii) an electron carrier or a combination of a coenzyme and 
an electron carrier, (iii) a stopping solution for the dehydrogenase 
reaction, (iv) a peroxidase and (v) a chromogen; and a reagent for use in 
the determination of dehydrogenase, comprising (i) a substrate of 
dehydrogenase, (ii) an electron carrier or a combination of a coenzyme and 
an electron carrier, (iii) a stopping solution for the hydrogenase 
reaction, (iv) a peroxidase and (v) a chromogen. 
In accordance with the method of the present invention, dehydrogenase or 
its substrate in the dehydrogenase reaction can be determined in an 
accurate and simple manner by utilizing the oxidative development of the 
chromogen by peroxidase. Unlike the conventional method, which has 
difficulty in determining the substrate in a high concentration, the 
method of the present invention provides an accurate determination of the 
substrate regardless of its concentration. Since the dye formed in the 
reaction of the present invention does not stick to tubes or cells of the 
determination device, unlike formazan dye, it becomes possible to treat a 
large quantity of samples continuously with an automatic analyzer. 
The terms "dehydrogenase" and "substrate" in the present invention mean 
such dehydrogenase and its substrate participating in the dehydrogenase 
reaction shown in the above diagrams 1 and 2, i.e. the reactions wherein 
the electrons from the substrate are transferred to O.sub.2 via the 
electron carrier or via the coenzyme and the electron carrier to form 
H.sub.2 O.sub.2. Typical examples of the dehydrogenase and its substrate 
are shown in Table 1. It is to be understood that the present invention is 
not limited to these combinations of the dehydrogenase and its substrate. 
Moreover, the substrate is not limited to those corresponding to each 
dehydrogenase but includes other substrates in so far as it can be 
specifically reacted with dehydrogenase. 
TABLE 1 
______________________________________ 
Substrate Dehydrogenase 
______________________________________ 
Glucose Glucose dehydrogenase 
(EC 1.1.1.47) 
Glucose-6-phosphate 
Glucose-6-phosphate 
dehydrogenase (EC 1.1.1.49) 
Malate Malate dehydrogenase 
(EC 1.1.1.37) 
3-.alpha.-Hydroxysteroid 
3-.alpha.-Hydroxysteroid 
dehydrogenase (EC 1.1.1.50) 
Lactate Lactate dehydrogenase 
(EC 1.1.1.27) 
L-Glutamate L-Glutamate dehydrogenase 
(EC 1.4.1.2) 
Leucine Leucine dehydrogenase 
(EC 1.4.1.9) 
Sarcosine Sarcosine dehydrogenase 
(EC 1.5.99.1) 
Amine Amine dehydrogenase 
(EC 1.4.99.3) 
Succinic acid Succinate dehydrogenase 
(EC 1.3.99.1) 
Choline Choline dehydrogenase 
(EC 1.1.99.1) 
Fructose Fructose dehydrogenase 
(EC 1.1.99.11) 
Sorbitol Sorbitol dehydrogenase 
(Japan. Pat. First Public. 
No. 2999/1981) 
______________________________________ 
The coenzyme employed in the method of the present invention includes 
NAD.sup.+, NADP.sup.+, flavins and the like. The flavins include flavin 
adenine dinucleotide (FAD), flavin mononucleotide (FMN) and the like. The 
dehydrogenation reaction involving the coenzyme includes those catalyzed 
by glucose dehydrogenase, glucose-6-phosphate dehydrogenase, malate 
dehydrogenase, 3-.alpha.-hydroxysteroid dehydrogenase, lactate 
dehydrogenase, L-glutamate dehydrogenase and leucine dehydrogenase among 
the dehydrogenases of the above Table 1. The concentration of the coenzyme 
is not particularly limited and is usually in the range of from 0.001 to 
2000 mM. 
Preferable electron carriers used in the method of the present invention 
include phenazine methosulfate (PMS), 1-methoxy-5-methylphenazium 
methylsulfate, 9-dimethylaminobenzo-.alpha.-phenazoxonium chloride and the 
like. These electron carriers may be used alone or in a mixture of more 
than one thereof. The concentration of the electron carrier in the 
reaction system is not specified but is usually in the range of from 
0.0001 to 1.0 mg/ml. 
The stopping solution for the hydrogenase reaction of the present invention 
includes an acid or an alkali, a buffer solution with different pH from 
that of the dehydrogenase reaction system, a dehydrogenase inhibitor, a 
surfactant, and the like. Suitable examples of the stopping solution are 
disclosed hereinafter. 
The chromogen of the present invention may be any compound which is 
oxidized to develop color with H.sub.2 O.sub.2 in the presence of 
peroxidase, including the following combinations: 
(1) 4-Aminoantipyrine/aniline derivatives 
(2) 4-Aminoantipyrine/phenol derivatives 
(3) Benzothiazolinone hydrazone derivatives/aniline derivatives. 
The aniline derivatives employed in the present invention include 
N-methyl-N-hydroxymethyl-3-methylaniline, 
N-ethyl-N-hydroxyethyl-3-methylaniline, 
N-ethyl-N-hydroxyethyl-3-ethylaniline, 
N-methyl-N-hydroxyethyl-3-methylaniline, 
N-methyl-N-hydroxypropyl-3-methylaniline, 
N-ethylN-hydroxypropyl-3-methylaniline, 
N-methyl-N-hydroxyethyl-3-ethylaniline, 
N-propyl-N-hydroxyethyl-3-methylaniline, 
N-methyl-N-hydroxyethyl-3-propylaniline, 
N,N-bis(.beta.-hydroxyethyl)-3-methylaniline, 
N,N-bis(.beta.-hydroxypropyl)-3-methylaniline, 
N,N-dimethyl-3-methylaniline, N,N-dimethyl-3-ethylaniline, 
N,N-dimethyl-3-propylaniline, N,N-diethyl-3-methylaniline, 
N,N-diethyl-3-ethylaniline, N,N-dipropyl-3-methylaniline, 
N-ethyl-N-(.beta.-methanesulfonamidethyl)-m-toluidine, 
N-ethyl-N-(.beta.-acetamidethyl)-3-methylaniline, N,N-dimethylaniline, 
N,N-diethylaniline, N,N-diisopropylaniline, 
N-methyl-N-hydroxyethylaniline, N-ethyl-N-hydroxyethylaniline, 
N-ethyl-N-sulfopropyl-m-toluidine, 
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, 
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, 
N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, 
3,5-dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, 
3,5-dimethyl-N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, 
N-ethyl-N-sulfopropyl-m-anisidine, 
N-ethyl-N-sulfopropyl-3,5-diemthylaniline, 
N-ethyl-N-sulfopropyl-3,5-dimethoxyanile, and the like. 
The phenol derivatives include p-chlorophenol, p-bromophenol, 
3,5-dichlorophenolsulfonic acid, 3-hydroxy-2,4,6-triiodobenzoic acid, and 
the like. 
The benzothiazolinone hydrazone derivatives include 
3-methyl-2-benzothiazolinone hydrazone and the like. 
The concentration of the aniline derivatives and the phenol derivatives is 
not specified but is most preferably in the range of from 0.1 to 20 mM. 
4-Aminoantipyrine is preferably used in a concentration of from about 1 to 
about 20 mM. The benzothiazolinone hydrazone derivatives are preferably 
used in a concentration of from about 0.1 to about 2 mM. 
As already mentioned hereinabove, the agent for determining substrate of 
dehydrogenase of the present invention comprises the first reagent 
containing (i) a dehydrogenase and (ii) an electron carrier or a 
combination of a coenzyme and an electron carrier, the second reagent 
containing (iii) a stopping solution for the dehydrogenase reaction, and 
the third reagent containing (iv) a peroxidase and (v) a chromogen. The 
second reagent and the third reagent may be mixed together before using. 
The agent for determining dehydrogenase of the present invention comprises 
the first reagent containing (i) a substrate of dehydrogenase and (ii) an 
electron carrier or a combination of a coenzyme and an electron carrier, 
the second reagent containing (iii) a stopping solution for the 
dehydrogenase reaction, and the third reagent containing (iv) a peroxidase 
and (v) a chromogen. The second reagent and the third reagent may be mixed 
together before using. 
For measuring the dehydrogenase or the substrate by the method of the 
present invention, the substrate, the dehydrogenase, the electron carrier 
and, if necessary, the coenzyme are mixed in the reaction mixture to carry 
out the dehydrogenase reaction. The electrons generated from the substrate 
are transferred to the coenzyme and the electron carrier (diagram 1) or to 
the electron carrier (diagram 2). The electrons are then transferred from 
the electron carrier to O.sub.2, which is converted into H.sub.2 O.sub.2 
via O.sub.2.sup.-. Then the reaction system is kept under fixed conditions 
and the dehydrogenase reaction is stopped. 
For stopping the hydrogenase reaction, the following methods may be 
employed but the present invention is not limited to these methods. 
1) METHOD BY ADDING AN ACID OR AN ALKALI 
Generally enzymes show a different stability depending on a pH value. That 
is, enzymes cannot exhibit their activities at a pH range higher or lower 
than a certain pH value. This property of enzymes suggests that the 
dehydrogenase reaction can be stopped by adding an acid or an alkali to 
the reaction system to alter the pH value of the reaction system so that 
the pH value falls into the pH range in which the enzymes cannot exhibit 
their activities. The acid or the alkali mentioned herein includes, for 
example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, 
nitric acid, sodium hydroxide, potassium hydroxide, aqueous ammonium, and 
the like, but the present invention is not limited to these acids and 
alkalis. 
2) METHOD BY ADDING A BUFFER SOLUTION WITH A DIFFERENT PH VALUE FROM THAT 
OF THE DEHYDROGENASE REACTION SYSTEM 
Alternatively, the pH value of the dehydrogenase reaction system may be 
altered in which the enzymes cannot exhibit their activities by mixing a 
buffer solution having a different pH value from that of the reaction 
system, instead of adding an acid or an alkali as above (1), and thereby 
the dehydrogenase reaction can be stopped. For example, when a 
dehydrogenase reaction is conducted in an acetate buffer solution (pH 4), 
a borate buffer (pH 10) is added to alter the pH value of the reaction 
system to pH 8, by which the dehydrogenase reaction is stopped. The buffer 
includes any conventional buffers so far as they can be employed in usual 
enzyme reactions. 
3) METHOD B Y ADDING A DEHYDROGENASE INHIBITOR TO THE REACTION SYSTEM 
As is well known, activities of enzymes are inhibited by various compounds, 
some of which can completely deactivate activate enzymes. By adding such 
inhibitors, the dehydrogenase reaction may also be ceased. For example, in 
case of sarcosine dehydrogenase, an addition of an aqueous 
p-chloromercurybenzoate leads to a complete deactivation of the 
dehydrogenase. Also in case of fructose dehydrogenase, activities of the 
enzyme can be competely inhibited by adding mercury chloride. The 
inhibitor is not limited to p-chloromercurybenzoate or mercury chloride 
but may be any conventional compounds so far as they can inhibit the 
dehydrogenase reaction and deactivate enzymes. 
4) METHOD BY ADDING A SURFACTANT 
Enzymes are proteins having a three dimensional steric structure, and when 
the enzymes lose this steric structure, they cannot show their activities. 
Therefore, by adding a substance which can destroy the steric structure of 
enzymes (i.e. dehydrogenase), it is possible to deactivate the 
dehydrogenase and to inhibit the dehydrogenase reaction so that the 
dehydrogenase reaction ceases. Such a substance capable of destroying the 
steric structure of dehydrogenase includes a surfactant such as sodium 
dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate and lithium dodecyl 
sulfate. The surfactant is not limited to the above exemplified 
surfactants, but includes any conventional surfactants which can 
deactivate the enzymes. 
After the reaction is stopped, the chromogen and peroxidase are added to 
the reaction system. The dye formed by the reaction with peroxidase is 
usually measured by an optical means employing a spectrophotometer. 
Since the dehydrogenation reaction is stopped in the method of the present 
invention, the reduced electron carrier (e.g. 1-mPMSH.sub.2) does not 
remained in the reaction system regardless of the substrate concentration. 
Therefore, in the method of the present invention, there are not such 
disadvantages that the electrons from the reduced electron carrier are 
transferred to the chromogen and thereby the chromogen is reduced and 
becomes incapable of color development, and that said electrons act on the 
dye formed from the chromogen and thereby decolorize the dye. This means 
that the method of the present invention can provide an accurate 
determination at any concentration of the substrate without inducing 
depression of the dye. Further, the dye formed by the method of the 
present invention, unlike the formazan dye, does not stick to tubes or 
cells of the determination device and thus enables a mass-treatment of 
test samples in a short time by employing an automatic analyzer. 
Furthermore, it is a great advantage that dehydrogenase or its substrate 
can be determined by the method of the present invention at a 
concentration of more than 0.005 M, which concentration could never be 
determined by the conventional method. 
The present invention is more specifically illustrated by the following 
Examples. However, it should be understood that the present invention is 
not limited to such Examples, but various changes and modifications can be 
made without departing from the scope and spirit of the invention. 
______________________________________ 
Example 1 (Determination of glucose-6-phosphate) 
______________________________________ 
Reaction components (1): 
Tris-hydrochloric acid buffer (pH 8.0) 
1.3 ml 
Glucose-6-phosphate dehydrogenase 
0.1 ml 
(EC 1.1.1.49) solution (0.5 U/ml) 
Glucose-6-phosphate solution (0-0.01 M) 
0.3 ml 
NAD.sup.+ solution 0.2 ml 
1-mPMS solution (0.5 mg/ml) 
0.1 ml 
Reaction components (2): 
4-Aminoantipyridine solution (10 mg/ml) 
0.3 ml 
TOOS solution (10 mg/ml) 
0.3 ml 
Peroxidase solution (100 U/ml) 
0.3 ml 
______________________________________ 
The above reaction components (1) (pH 8.0) were mixed and reacted at 
37.degree. C. for just 5 minutes. Thereto an aqueous SDS solution (300 
mg/ml) was added to stop the dehydrogenase reaction. Then the above 
reaction components (2) were added and the mixture was reacted for further 
4 minutes, and then the absorbance of the reaction mixture was measured at 
550 nm. FIG. 2 shows a relationship between the concentrations of the 
substrate glucose-6-phosphate and the OD values after the reaction 
(obtained by deduction of blank value from the found value; designated as 
.DELTA.OD.sub.550). 
REFERENCE EXAMPLE 
The dehydrogenation reaction of Example 1 was repeated in the same reaction 
system as the reaction components (1) except that 1.4 ml of 
Tris-hydrochloric acid buffer was employed. After the reaction for just 5 
minutes, the reaction components (2) (the same as in Example 1) were 
immediately added to the mixture, and the mixture was reacted for 4 
minutes. The absorbance of the reaction mixture was measured at 550 nm, 
results of which are shown in FIG. 2. 
As shown in FIG. 2, it is clear that the accurate determination is possible 
by the method of the present invention even if the substrate concentration 
is at a high level. On the contrary, the conventional method can determine 
the substrate only at a low level of the substrate but cannot determine 
the substrate at a high level (more than about 0.005 M) due to color 
depression leading to the absorbance with no significant difference from 
that at the substrate concentration of 0 M. 
______________________________________ 
Example 2 
(Determination of activity of sarcosine dehydrogenase) 
Reaction components (1): 
______________________________________ 
Tris-hydrochloric acid buffer (pH 8.0) 
1.4 ml 
Sarcosine dehydrogenase (EC 1.5.99.1) 
0.1 ml 
solution 
Sarcosine solution (1 M) 
0.1 ml 
1-mPMS solution (0.5 mg/ml) 
0.1 ml 
______________________________________ 
The above reaction components (1) (pH 8.0) were mixed and reacted at 
37.degree. C. for 1 minutes. Thereto an aqueous p-chloromercurybenzoate 
(0.1 M, 0.1 ml) was added to stop the dehydrogenase reaction. Then the 
reaction components (2) (the same as in Example 1) were added to the 
mixture. The reaction was conducted for 4 minutes and then the absorbance 
of the reaction mixture was measured at 550 nm. The above procedures were 
repeated except that the reaction was conducted for 2, 3, 4, 5, 6 and 7 
minutes. FIG. 3 shows a relationship between the reaction time and the OD 
values (obtained by deduction of blank value from the found value; 
designated as .DELTA.OD.sub.550). As is clear from FIG. 3, the enzyme 
activity of sarcosine dehydrogenase is accurately determined by the method 
of the present invention. 
______________________________________ 
Example 3 
(Determination of activity of fructose dehydrogenase) 
______________________________________ 
Reagent 1: 
Fructose 1 M 
m-PMS 10 mM 
4-AA 1 mg/ml 
McIlvaine's buffer solution (pH 4.5) 
Reagent 2 
TOOS 1 mg/ml 
TES 
(N-tris(hydroxymethyl)methyl-2- 
aminoethanesulfonic acid) 
buffer solution (pH 7.5) 
Reagent 3 
Sodium dodecyl sulfate (SDS) 
10% 
______________________________________ 
Fructose dehydrogenase solutions (0-20 ml) were subjected to fructose 
dehydrogenase measurement employing the above reagents 1 to 3. 
First, the reagent 1 was taken into a test tube and pre-incubated at 
37.degree. C. Thereto a fructose dehydrogenase solution was added and 
reacted at 37.degree. C. for 5 minutes. After the reaction, the reagent 3 
was added to the reaction mixture to stop the reaction and then the 
reagent 2 (1 ml) was added. Five minutes after the addition of the reagent 
2, the absorbance of the reaction mixture was measured at 550 nm with 
control of blank. As a result, as shown in FIG. 4, the graph was a 
straight line. Thus, it was proved that the fructose dehydrogenase 
activity was correctly measured.