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A Rapid and Sensitive Colorimetric Assay for Determination of Adenosine Kinase Activity

A technology of adenosine kinase and colorimetry, which is applied in the field of colorimetry for rapid and sensitive determination of adenosine kinase activity, can solve the problems of harmfulness to human body and inability to accurately reflect the initial rate of ADK catalytic reaction, and achieve simple steps and short detection time. short effect

Inactive Publication Date: 2019-11-26
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, radionucleoside substrates are harmful to humans and cannot accurately reflect the initial rate of ADK-catalyzed reactions

Method used

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  • A Rapid and Sensitive Colorimetric Assay for Determination of Adenosine Kinase Activity
  • A Rapid and Sensitive Colorimetric Assay for Determination of Adenosine Kinase Activity
  • A Rapid and Sensitive Colorimetric Assay for Determination of Adenosine Kinase Activity

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Embodiment 1

[0021] Preparation and concentration determination of embodiment 1, BmADK

[0022] The BmADK gene was cloned from silkworm cDNA, and the cloning primers were as follows: forward primer 5'-cgcggatccatggacgtttctgattccatatgtg-3' (SEQ ID NO.1); reverse primer 5'-cccaagctttcagtcattgtattcgctgggtc-3' (SEQ ID NO.2). After digestion with BamH I and Hind III, it was inserted into pSKB2 expression vector, expressed in Escherichia coli at 25°C, and purified by nickel column affinity chromatography and gel filtration chromatography. The BmADK concentration was determined using a NanoDrop 2000C spectrophotometer (ThermoFisher, USA). After 12.5% ​​SDS-PAGE and Coomassie blue staining, it was found that the purity of BmADK reached 99%, and the final concentration of BmADK after purification was 3.68 mg / mL.

Embodiment 2

[0023] Example 2, ADK activity analysis optimization

[0024] With ATP and adenosine as substrates, the hydrogen ions generated by the ADK-catalyzed reaction change the pH value of the reaction system, resulting in a color change of the reaction system, which can be easily represented by the change in absorbance. The change in absorbance is proportional to the concentration of hydrogen ions. Therefore, ADK activity and initial reaction speed can be determined according to the change in absorbance, and the reaction principle is as follows: figure 1 shown.

[0025] In order to determine the maximum absorption wavelength of the reaction system, the pH value of the system was changed with hydrochloric acid, and the wavelength scanning of the reaction system was carried out. A serial dilution of hydrochloric acid (10 μL) was added to the reaction system (990 μL), and mixed well. After equilibrating for 3 minutes, a full-wavelength scan of 450-800 nm was performed on each sample,...

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Abstract

The invention relates to a colorimetric method capable of rapidly and sensitively determining adenosine kinase (ADK) activity. The method comprises the following steps: taking ATP (Adenosine Triphosphate) and adenosine as a substrate, taking bromothymol blue as an indicator, taking magnesium acetate and glycine-sodium hydroxide as a buffer solution, detecting the absorbance with a to-be-determinedsolution containing adenosine kinase under the condition of 614nm. BVy utilizing the bromothymol blue as the pH indicator, the reaction system has a maximum absorption peak at 614nm, and the change value of the absorbance is in positive correlation to hydrogen ions produced by ADK catalysis. The method for detecting the adenosine kinase has the advantages of being rapid, simple, convenient, reliable, sensitive and economic, and the traditional adenosine kinase activity analysis method can be well replaced.

Description

technical field [0001] The invention belongs to the field of enzyme activity detection and relates to a colorimetric method for rapidly and sensitively measuring adenosine kinase activity. Background technique [0002] Adenosine kinase (ADK; adenosine 5'-phosphotransferase, EC 2.7.1.20), was first isolated in yeast cells by Kornberg in 1951. ADK belongs to the ribokinase family and is one of the most abundant nucleoside kinases in mammalian tissues. As the first enzyme in the purine salvage pathway, ADK can catalyze the transfer of the γ-phosphate group of adenosine triphosphate (ATP) to adenosine to generate AMP (Adenosine+ATP→AMP+ADP). ADK can effectively protect tissues such as the heart and nerves by regulating the concentration of intracellular and extracellular adenosine. In a diabetic model, reducing ADK activity inhibited T-lymphocyte proliferation. ADK also plays an important role in promoting intracellular methylation, knocking out ADK (ADK - / - ) in transgenic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 王叶菁李瑜何华伟周海梦赵萍夏庆友
Owner SOUTHWEST UNIV