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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (3): 487-492. doi: 10.19723/j.issn.1671-167X.2019.03.016

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Fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity in human blood samples

Jia-yu WANG,Mei-ping ZHAO△()   

  1. Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
  • Received:2019-03-13 Online:2019-05-22 Published:2019-06-26
  • Supported by:
    Supported by the National Natural Science Foundation of China (81571130100), the Fundamental Research Funds for the Central Universities: Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2017MC008), Innovation Fund of Sunan Institute for Molecular Engineering, Peking University (SIM2018002848)

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Abstract: Objective: To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples.Methods: An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT(BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T* GAATCTCTCTAG*T*C*T-3'[“X” represents AP site; The phosphorothioated nucleotides (at 3’ side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples. Results: With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3).Conclusion: The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.

Key words: Spectrometry, fluorescence, Apurinic/apyrimidinic endonuclease 1, DNA probes, Serum, Peripheral blood mononuclear cells

CLC Number: 

  • R446.1

Figure 1

A, Fluorescence responses of the APE1 probe (200 nmol/L) to APE1 (2.0 U/mL) in different reaction buffers. NEB buffer 4+0.04% Triton X-100 is the new reaction buffer developed in this work; B, Fluorescence responses of the APE1 probe (300 nmol/L) to APE1 at different concentrations in the range from 0.01 to 2.0 U/mL in the new reaction buffer; C, Fluorescence responses of the APE1 probe (300 nmol/L) to APE1 at different concentrations in the range from 0.005 to 0.1 U/mL in the new reaction buffer; D, Linear working range of the APE1 probe in the new reaction buffer"

Figure 2

A, Isolation of PBMCs by density gradient centrifugation; B, Comparison of the APE1 concentration in human serum determined by different dilution folds PBMC, peripheral blood mononuclear cells."

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