Measurement of phenoloxidase activity

doi:10.1038/nprot.2007.468

Method Article

Measurement of phenoloxidase activity

https://doi.org/10.1038/nprot.2007.468

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Biochemistry

Computational biology and bioinformatics

prophenoloxidase (PPO)

phenoloxidase (PO)

4-methylcatechol

In the chelicerate, hemocyanin is the functional substitution of prophenoloxidase (PPO). Upon activation, hemocyanin is converted to phenoloxidase (PO), which oxidizes the phenolic substrate such as 4-methylcatechol to produce quinone product, which has a maximum absorbance at 405 nm.

PO activity was measured as described¹, with modifications.

Incubate purified hemocyanin with proteases in 50 mM Tris-HCl, pH 8.3, containing 0.05 M NaCl, at 20 ˚C for 10 min.
Add PAMPs (pathogen-associated molecular patterns eg: LPS or LTA) dissolved in the same buffer.
Then add 1 mM 4ME in 0.1 M potassium phosphate, pH 6.0.
Immediately after the addition of the substrate, continuously monitor the absorbance at 405 nm using a microplate reader (Molecular Devices, USA).
The PO activity is presented as A405 at 5 min after the addition of substrate.

The PO activity varies depending on the type of microbial proteases and PAMPs which are used for the activation. The typical range of PO activity at A405 is 0.1-0.8.

Jiang, H., Wang, Y. & Kanost, M. R. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc Natl Acad Sci U S A. 95, 12220-12225 (1998).

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Measurement of phenoloxidase activity

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