Method for the colorimetric determination of an analyte by means of benzyl alcohol dehydrogenase and a chromogenic redox indicator
First Claim
1. A method for the colorimetric determination of NAD(P)H or an analyte which forms NAD(P)H, comprising the steps ofa) reacting NAD(P)H with benzyl alcohol dehydrogenase in the presence of a nitrosoaniline compound of the formula I:
- ##STR45## wherein said nitrosoaniline compound is an electron acceptor, b) enzymatically reducing the nitrosoaniline compound to a colored quinone diimiine, andc) measuring any color produced as an indication of the amount of NAD(P)H,whereinR1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, andR is a residue which produces a color by electron conjugation with a quinone diimiine system.
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Abstract
The invention concerns a method for the colorimetric determination of NADH by means of enzymatic oxidation with benzyl alcohol dehydrogenase whereby an aromatic nitrosoaniline compound of the general formula I is reduced to a colored quinone diimine ##STR1## in which R1 denotes hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkyl or alkylene substituted if desired by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, amino substituted if desired once or twice by alkyl which in turn can be substituted if desired by hydroxy, PO3 H2, SO3 H or carboxy and R denotes a residue which forms a color by electron conjugation with a quinone diimiine system. The invention also concerns a corresponding agent as well as a chromogenic nitroso compound of formula I as well as its use for the enzymatic colorimetric determination of an analyte.
83 Citations
26 Claims
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1. A method for the colorimetric determination of NAD(P)H or an analyte which forms NAD(P)H, comprising the steps of
a) reacting NAD(P)H with benzyl alcohol dehydrogenase in the presence of a nitrosoaniline compound of the formula I: - ##STR45## wherein said nitrosoaniline compound is an electron acceptor, b) enzymatically reducing the nitrosoaniline compound to a colored quinone diimiine, and
c) measuring any color produced as an indication of the amount of NAD(P)H, wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and R is a residue which produces a color by electron conjugation with a quinone diimiine system. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
- ##STR45## wherein said nitrosoaniline compound is an electron acceptor, b) enzymatically reducing the nitrosoaniline compound to a colored quinone diimiine, and
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13. A nitrosoaniline compound of the formula I, ##STR54## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, an alkyl or alkylene which is unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino which is unsubstituted or substituted once or twice by an alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which produces a color by electron conjugation with a quinone diimiine system.
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22. A reagent for the colorimetric determination of NAD(P)H comprising benzyl alcohol dehydrogenase and a nitrosoaniline compound of formula I:
- ##STR62## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.
- ##STR62## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
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23. A reagent for the colorimetric determination of benzyl alcohol dehydrogenase comprising NAD(P)H and a direct chromogenic electron acceptor, wherein the electron acceptor is a nitrosoaniline compound of formula I:
- ##STR63## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.
- ##STR63## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
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24. A reagent for the colorimetric determination of a NAD(p)+ -dependent dehydrogenase comprising a substrate of the dehydrogenase to be determined, NAD(P)+, an NAD(P)H-converting dehydrogenase and a direct chromogenic electron acceptor,
wherein the NAD(P)H-converting dehydrogenase is benzyl alcohol dehydrogenase and wherein the electron acceptor is a nitrosoaniline compound of formula I: - ##STR64## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.
- ##STR64## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
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25. A reagent for the colorimetric determination of an analyte comprising an analyte-specific NAD(P)-dependent dehydrogenase, NAD(P), an NAD(P)H-oxidizing enzyme and a direct chromogenic electron acceptor, wherein said NAD(P)H-oxidizing enzyme is benzyl alcohol dehydrogenase and said direct chromogenic electron acceptor is a nitrosoaniline compound of formula I:
- ##STR65## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system.
- ##STR65## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
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26. A process for the production of a nitrosoaniline compound of formula I:
- ##STR66## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
R is a residue which forms a color by electron conjugation with a quinone diimiine system, comprising reacting a p-nitrosophenyl ether of formula XVII;
##STR67## with an amino compound of formula XVIII;
##STR68##
- ##STR66## wherein R1 is selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, aryloxy, alkylthio or arylthio, alkyl or alkylene unsubstituted or substituted by carboxy, PO3 H2, dialkylphosphinyl or SO3 H, and an amino unsubstituted or substituted once or twice by alkyl which in turn can be unsubstituted or substituted by hydroxy, PO3 H2, SO3 H or carboxy, and
Specification