Method for detecting fluorescence or absorbance, method for suppressing background, method for measuring adp, method for measuring activity of adp-synthesizing enzyme, and method for measuring activity of glucosyltransferase

Patent metadata

Field	Value
Publication number	US-2016289728-A1
Application number	US-201415037814-A
Country	US
Kind code	A1
Filing date	Nov 21, 2014
Priority date	Nov 21, 2013
Publication date	Oct 6, 2016
Grant date	—

Abstract

In a method for detecting fluorescence or absorbance of the present invention, a diaphorase causes reduction from resazurin to resorufin in the presence of an SH reagent and NADH or NADPH, and the resulting fluorescence intensity or absorbance is measured. A method for measuring ADP of the present invention includes a 2-1 process in which glucose is reacted with ADP and an ADP-dependent hexokinase, a 2-2 process in which the glucose-6-phosphate obtained in the 2-1 process is reacted with NAD or NADP and glucose-6-phosphate dehydrogenase, and a 2-3 process in which resazurin is reacted with the NADH or NADPH obtained in the 2-2 process and a diaphorase in the presence of an SH reagent, and the resulting fluorescence intensity or absorbance is measured.

First claim

1 . A method for detecting fluorescence or absorbance comprising: reducing, by a diaphorase, resazurin to resorufin, in the presence of an SH reagent and NADH or NADPH, and measuring the resulting fluorescence intensity or absorbance, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

2 . The method for detecting fluorescence or absorbance according to claim 1 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide or N-(2-sulfoethyl)maleimide.

3 . A method for suppressing background comprising: an operation in which, when a fluorescence intensity or absorbance caused by reactions of NADH or NADPH, resazurin and a diaphorase in the presence of a reducing agent is measured, the reactions are caused in the presence of an SH reagent, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

4 . The method for suppressing background according to claim 3 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide, or N-(2-sulfoethyl)maleimide.

5 . A method for measuring ADP comprising: a 2-1 process in which glucose is reacted with ADP and an ADP-dependent hexokinase to produce glucose-6-phosphate; a 2-2 process in which the glucose-6-phosphate obtained in the 2-1 process is reacted with NAD or NADP and glucose-6-phosphate dehydrogenase to produce NADH or NADPH; and a 2-3 process in which resazurin is reacted with the NADH or NADPH obtained in the 2-2 process and a diaphorase in the presence of an SH reagent, and the resulting fluorescence intensity or absorbance is measured, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

6 . (canceled)

7 . The method for measuring ADP according to claim 5 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide or N-(2-sulfoethyl)maleimide.

8 . A method for measuring activities of ADP-producing enzymes comprising: a 1-1 process in which an ADP-producing enzyme is reacted with a substrate in the presence of ATP to convert the ATP into ADP; a 2-1 process in which glucose is reacted with the ADP obtained in the 1-1 process and an ADP-dependent hexokinase to produce glucose-6-phosphate; a 2-2 process in which the glucose-6-phosphate obtained in the 2-1 process is reacted with NAD or NADP and glucose-6-phosphate dehydrogenase to produce NADH or NADPH; and a 2-3 process in which resazurin is reacted with the NADH or NADPH obtained in the 2-2 process and a diaphorase in the presence of an SH reagent, and the resulting fluorescence intensity or absorbance is measured, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

9 . (canceled)

10 . The method for measuring activities of ADP-producing enzymes according to claim 8 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide or N-(2-sulfoethyl)maleimide.

11 . The method for measuring activities of ADP-producing enzymes according to claim 8 , wherein the ADP-producing enzyme is at least one type selected from the group including kinases, ATPases, nitrogenases, tetrahydrofolate synthases, acetyl-CoA carboxylase, pyruvate carboxylase, and glutathione synthase.

12 . A method for measuring activities of a glycosyltransferase, the method comprising: a first process in which GDP or UDP produced during a glycosyltransferase reaction is reacted with an NDP kinase in the presence of ATP, or CMP produced during the glycosyltransferase reaction is reacted with NMP kinase or a CMP kinase in the presence of ATP, and thus ADP corresponding to an amount of the GDP, UDP or CMP is produced; a 2-1 process in which glucose is reacted with the ADP obtained in the first process and an ADP-dependent hexokinase to produce glucose-6-phosphate; a 2-2 process in which the glucose-6-phosphate obtained in the 2-1 process is reacted with NAD or NADP and glucose-6-phosphate dehydrogenase to produce NADH or NADPH; and a 2-3 process in which reactions of the NADH or NADPH obtained in the 2-2 process and a diaphorase are caused in the presence of an SH reagent, and the resulting fluorescence intensity or absorbance is measured, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

13 . (canceled)

14 . The method for measuring activities of a glycosyltransferase according to claim 12 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide or N-(2-sulfoethyl)maleimide.

15 . The method for measuring activities of a glycosyltransferase according to claim 12 , wherein the glycosyltransferase is at least one type selected from the group including fucosyltransferases, mannosyltransferases, glucosyltransferases, galactosyltransferases, glucuronosyltransferases, xylosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, and sialyltransferases.

16 . An ADP measurement kit comprising glucose, an ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, a diaphorase, NAD and/or NADP, resazurin and an SH reagent, wherein the SH reagent is a maleimide compound represented by the following General Formula [1] or 2-iodoacetamide,

(where, in Formula [1], R 1 represents a hydrogen atom, a linear or branched alkyl group that has 1 to 6 carbon atoms, or a linear or branched sulfoalkyl group that has 1 to 6 carbon atoms).

17 . (canceled)

18 . The ADP measurement kit according to claim 16 , wherein the maleimide compound represented by General Formula [1] is N-ethylmaleimide, maleimide or N-(2-sulfoethyl)maleimide.

19 . An ADP-producing enzyme activity measurement kit comprising glucose, an ADP-dependent hexokinase, glucose-6-phosphate dehydrogenase, a diaphorase, NAD and

Assignees

• Univ Tokyo

Inventors

• Kumagai Kazuo
• Okabe Takayoshi
• Kojima Hirotatsu
• Nagano Tetsuo

Classifications

• C12Q1/42

  involving phosphatase · CPC title

• G01N2333/91091

  Glycosyltransferases (2.4) · CPC title

• C12Q1/008 Primary

  for determining co-enzymes or co-factors, e.g. NAD, ATP · CPC title

• C12Q1/48

  involving transferase · CPC title

• C12Q1/32

  involving dehydrogenase · CPC title