Measurement of phenoloxidase activity
Lab/Group: Ding Lab (National University of Singapore)
Related Journal & Article Information
Journal: Nature Immunology
Introduction
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.
Materials
Reagents
Equipment
Procedure
PO activity was measured as described1, with modifications.
1. Incubate purified hemocyanin with proteases in 50 mM Tris-HCl, pH 8.3, containing 0.05 M NaCl, at 20 ˚C for 10 min.
2. Add PAMPs (pathogen-associated molecular patterns eg: LPS or LTA) dissolved in the same buffer.
3. Then add 1 mM 4ME in 0.1 M potassium phosphate, pH 6.0.
4. Immediately after the addition of the substrate, continuously monitor the absorbance at 405 nm using a microplate reader (Molecular Devices, USA).
5. The PO activity is presented as A405 at 5 min after the addition of substrate.
Troubleshooting
Critical Steps
Anticipated Results
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.
References
1. 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).
Acknowledgements
Keywords
prophenoloxidase (PPO); phenoloxidase (PO); 4-methylcatechol
Figure 1
Activation of HMC/PPO to PO by microbial proteases and PAMPs
PO activity triggered from hemocyanin by microbial proteases such as subtilisin (column 2) and Pseudomonas elastase (column 6). PAMP molecules such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS) further enhances the PPO activation induced by microbial proteases, in a dose-dependent manner.
