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Development
of Coupling Reagents for Enzymatic Determination of Clinical Interests
Makoto Mizoguchi
Research section in Dojindo Labs
Enzymatic methods
for determining biogenic compounds are a key technology in quantification
of various analites of clinical interests in biomatrix and have found
wide applications in clinical diagnosis due to their high specificity
and rapidity.
The enzymes employed for this purpose primarily include oxidases and
dehydrogenases. As shown in Fig. 1, hydrogen peroxide derived from
a oxidase reaction with its substrate is used for enzymatic oxidation
of oxidative chromogenic reagents.
Concentration of resulting dye indicates the amounts of the substrate
can be measured spectrophotometorically.
The typical reaction scheme for quantification of biogenic compounds
using 4-aminoantipyrine (4-AA) is shown in Fig. 2B. The aniline derivatives
(hydrogen donor) and 4-AA as a coupling reagent are condensed by hydrogen
peroxide in presence of peroxidase (POD).
Over several decades, a number of hydrogen donors have been developed
and modified. However, only a few coupling reagents have been developed,
such as 3-methyl-2-benzothiazolinonehydrazone (MBTH). As most of amine
compounds were unstable and less reactive, they could not be utilized
as coupling reagents.
The required properties for the oxidative reagent are as follows;
- 1) a long
wavelength of absorption maximum of resulting dye.
- 2) high
molar absoptivity of resulting dye.
- 3) high
water solubility.
- 4) stability
in an aqueous solution.
The reactions of oxidative chromogenic reagents is interfered
by several compounds in biological samples. Bilirubin and ascorbic
acid are most troublesome materials in serum and urine for oxidative
chromogenic reaction. In order to overcome these problems, the
development of coupling reagents was necessary.
In the effort to develop the compounds as coupling reagents,
we recently developed NCP-04, 05,06 and 07 as shown in Fig.
2A. These compounds were reacted with hydrogen donors by hydrogen
peroxide in presence of POD and produced green colored dyes.
The wavelength of absoptin maxima of resulting dyes by NCP compounds
shift 200 nm longer in wavelength compare with resulting dyes
by 4-AA. (Fig.3 , Table 1)
Table 1 lambdamax of chromogen formed by oxidative condensation
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| Trinder's
reagents |
NCP-04 |
NCP-05 |
NCP-06 |
NCP-07 |
| ADOS |
723.4
nm |
742.2
nm |
727.2
nm |
727.6
nm |
| ADPS |
720.8
nm |
740.4
nm |
723.8
nm |
724.7
nm |
| ALPS |
741.6
nm |
755.4
nm |
737.0
nm |
737.6
nm |
| DAPS |
729.0
nm |
753.0
nm |
736.0
nm |
738.8
nm |
| DAOS |
735.2
nm |
758.3
nm |
741.6
nm |
741.3
nm |
| TOOS |
749.6
nm |
753.4
nm |
750.6
nm |
750.8
nm |
| MAOS |
780.4
nm |
786.2
nm |
781.2
nm |
782.0
nm |
| MAPS |
771.4
nm |
783.2
nm |
777.2
nm |
778.0
nm |
In the sensitivity on determination of hydrogen peroxide, NCP
derivatives was 1.5 - 2.8 times than 4-AA. (Fig.3, 4)
-
-
-
The oxidative condensation of the Trinder's reagents with NCP
derivatives was not affected with pH over the range from 4 to
8. Aqueous solution of NCP derivatives was stable over 25 days,
and their mixed solutions with TOOS were also stable at 4C for
25 days except for NCP-04 and 05. (Table 2)
-
- Table
2 Stability of NCP derivatives
-
|
Absorbance
at 750 nm after 25 days |
| Reagent |
Aqueous
solution
at 30C |
Aqueous
solution containing
100uM TOOS at 4C |
| NCP-04 |
0.009 |
0.147 |
| NCP-05 |
0.001 |
0.095 |
| NCP-06 |
0.000 |
0.002 |
| NCP-07 |
0.001 |
0.005 |
It is suggested that these stability of NCP-05 and 06 is caused
by introduced methoxy groups. The interferences of bilirubin
was least on coupling reaction of NCP-06 and DAPS. (Fig.5)
-
In Fig. 5, minimum interference of bilirubin was observed in
the combination of NCP-06 and DAPS. The reason is not clear,
but It might be in the stability of resulted dye. NCP derivatives
were useful for highly sensitive determination of hydrogen peroxide
derived from enzymatic reaction, such as glucose oxidase.
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