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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

An activity measurement and detection method technology, which is applied in the fields of fluorescence or absorbance detection, background suppression, ADP measurement, activity measurement of ADP-generating enzymes, and glycosyltransferase activity measurement, which can solve difficult analysis and detection sensitivity. Low, complicated operation and other problems

Inactive Publication Date: 2016-07-13
THE UNIV OF TOKYO
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Problems solved by technology

[0008] However, the above method requires the chemical synthesis of the labeled sugar donor, the determination requires special equipment (liquid scintillator, LC-MS), and it is difficult to perform simple analysis, especially difficult to use microplates for a large number of quantitative analysis (Qualcomm volume screening)
[0009] In addition, it is a cost-intensive method in terms of fees
[0010] On the other hand, it is known to use a commercially available analysis kit (Transcreener (registered trademark) GDPAssay, UDP2Assay, AMP2 / GMP2Assay manufactured by Bellbrook Labs, Inc.) to detect the protein produced by the glycosyltransferase reaction by the fluorescence polarization method. The method of quantifying nucleotides, the method of measuring the absorbance of NADH by enzymatic coupling reaction (non-patent literature 5, 6), or the method of quantifying by the color development of malachite green by freeing phosphate (non-patent literature 7), but the detection sensitivity is low
[0011] In addition, there is also known a method of measuring by enzyme coupling to induce chemiluminescence of luciferase (Patent Document 1), but the operation is complicated and the sensitivity is not clear
[0012] In addition, recently, a high-speed analysis method for the transfer of fluorescently labeled sugars using the fluorescence polarization method has been reported (Non-Patent Document 8), but it can only be used for high-molecular substrates and cannot be applied to all sugars. base transferase
[0013] As such, it is difficult for existing methods to analyze all glycosyltransferases easily and with high sensitivity using microplates, and it is difficult to perform high-throughput screening of glycosyltransferase activity modulators

Method used

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  • 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
  • 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
  • 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

Examples

Experimental program
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Effect test

Embodiment 1

[0296] Use the calibration curve of the GDP of the method of the present invention, UDP

[0297] Calibration curves of GDP and UDP were created using the reactions of the first step and the second step of the present invention.

[0298] As materials, GDP (Wako Pure Chemical Industries, Ltd. catalog number 078-04741), UDP (Wako Pure Chemical Industries, Ltd. catalog number 212-00861), ATP (Wako Pure Chemical Industries, Ltd. catalog number 18-16911), NDP kinase from baker's yeast (Sigma-Aldrich (Sigma-Aldrich) catalog number N0379), glucose (Wako Pure Chemical Industries, Ltd. catalog number 045-31162), ADP-dependent hexokinase (Asahi Kasei Pharmaceutical Co., Ltd. Farm company) catalog number T-93, from thermococcus (Thermococcuslitoralis)), G-6-P dehydrogenase (Oriental yeast industry company (Oriental yeast industry company) catalog number 46857003), diaphorase (especially Nichiko (Unichika Corporation) catalog number B1D111), NADP (Oriental Yeast Industry Corporation (Orie...

Embodiment 2

[0309] Calibration curve for CMP using the method of the invention

[0310] A calibration curve of CMP was created using the reactions of the first step and the second step of the present invention.

[0311] As materials, CMP (Wako Pure Chemical Industries, Ltd. catalog number 034-05361), CMP kinase (human CMPK1, Prospec catalog number PKA-002), DTT (Wako Pure Chemical Industries, Ltd. No. 040-29223), N-ethylmaleimide (Wako Pure Chemical Industries, Ltd. Catalog No. 054-02061). Dissolve CMP in buffer F (100 mM Tris-HCl (pH9), 13.5 mM MgCl 2 , 150 mM KCl, 0.1% Triton X-100 (TritonX-100)) to prepare a 0-40 μM solution. Prepare the mixed solution for enzyme coupling according to the following composition.

[0312] Since CMP kinase requires a reducing agent, DTT was used as a reducing agent in the first step, and N-ethylmaleimide was used as an SH reagent for inactivation in the second step.

[0313] Mixture D for the first step

[0314]

[0315] Mixture E for the second s...

Embodiment 3

[0319] Time-dependent changes in fluorochrome

[0320] After performing the reaction in the same manner as in Example 1 using 40 μM of GDP or UDP, the plate was taken out at intervals only during the reaction in the second step for fluorescence measurement. In addition, using 40 μM CMP, the reaction was carried out in the same manner as in Example 2, and only in the reaction in the second step, the plate was taken out at intervals for fluorescence measurement. The results are expressed in Figure 4 . It can be seen from this that, in the case of quantitative GDP or UDP, the reaction of the second step is completed within 25 minutes, and the fluorescence value is kept stably for at least 120 minutes afterwards; in the case of quantitative CDP, the reaction of the second step is completed within 25 minutes. At the end of the minute, there was little change in the fluorescence value until at least 120 minutes after 60 minutes.

[0321] Therefore, in the case of quantitative GD...

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Abstract

A method for detecting fluorescence or absorbance according to the present invention is characterized by comprising reducing resazurin into resorufin by a diaphorase in the presence of an SH reagent and NADH or NADPH and then measuring the resultant fluorescence intensity or absorbance. A method for measuring ADP according to the present invention is characterized by comprising: step (2-1) for treating glucose with ADP and ADP-dependent hexokinase; step (2-2) for treating glucose-6-phosphate obtained in the above step (2-1) with NAD or NADP and glucose-6-phosphate dehydrogenase; and step (2-3) for treating resazurin with NADH or NADPH obtained in the above step (2-2) and a diaphorase in the presence of an SH reagent and then measuring the resultant fluorescence intensity or absorbance.

Description

technical field [0001] The present invention relates to a method for detecting fluorescence or absorbance capable of highly sensitively measuring the fluorescence intensity or absorbance generated by the reduction of resazurin to resorufin, a method for background suppression, a method for measuring ADP, and a simple and highly sensitive method for measuring kinases A method for the activity of an ADP-generating enzyme or glycosyltransferase, a method for screening an activity modulator of an ADP-generating enzyme such as a glycosyltransferase or a kinase using the method, and a measurement kit used in the method. [0002] This application claims priority based on Japanese Patent Application No. 2013-240926 for which it applied in Japan on November 21, 2013, and uses the content here. Background technique [0003] Glycosyltransferase is a general term for enzymes that catalyze a reaction in which a glycosyl group is transferred from a glycosyl-containing donor (G) to an acce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12Q1/32C12Q1/48G01N21/78
CPCC12Q1/008C12Q1/32G01N2333/90209G01N2333/91097G01N2333/91215C12Q1/42C12Q1/48C12Q1/485G01N2333/91091
Inventor 熊谷和夫冈部隆义小岛宏建长野哲雄
Owner THE UNIV OF TOKYO
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