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Application of methyl parathion hydrolase (MPH) as a labeling enzyme

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Abstract

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 ± 13.19 s−1, which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody–enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [–(Gly–Ser)5–] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

The 3-D structure of MPH. (a) monomer showing C- and N-terminals; (b) the crystal structure of the dimer

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Acknowledgement

This work was supported by 863 project (No. 2006AA020904).

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Authors and Affiliations

  1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China

    Wei Yang, Ya-Feng Zhou, Zhi-Ping Zhang, Yan Leng & Xian-En Zhang

  2. Graduate School, Chinese Academy of Sciences, Beijing, 100049, China

    Wei Yang

  3. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China

    He-Ping Dai & Xiao-Hua Zhang

  4. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

    Li-Jun Bi

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  1. Wei Yang
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  2. Ya-Feng Zhou
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  6. Xiao-Hua Zhang
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Correspondence to Xian-En Zhang.

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W. Yang and Y.-F. Zhou contributed equally to this work.

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Yang, W., Zhou, YF., Dai, HP. et al. Application of methyl parathion hydrolase (MPH) as a labeling enzyme. Anal Bioanal Chem 390, 2133–2140 (2008). https://doi.org/10.1007/s00216-008-1987-y

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  • DOI: https://doi.org/10.1007/s00216-008-1987-y

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