Non-specific electrostatic interactions and the conversion of horseshoe crab haemocyanin into a phenoloxidase.

Haemocyanin is a haemolymph (blood)-based protein and the functional equivalent to haemoglobin - supplying tissues with oxygen in decapod crustaceans, chelicerates, and shelled molluscs. In addition to oxygen transport, haemocyanin plays several roles in innate immunity, wound healing, and ecdysis. Under certain conditions in vitro and in vivo, horseshoe crab (Limulus polyphemus) haemocyanin is converted into a phenoloxidase-like enzyme, yet the protein-ligand interactions associated with this conversion remain unclear. Negatively charged ligands, such as phosphatidylserine and sodium dodecyl sulphate, represent effective endogenous and exogenous activators, respectively. Herein, we explored the nature of the interaction between haemocyanin and phosphatidylserine. We used several spectroscopic techniques and phenoloxidase assays to follow the electrostatic interactions. Manipulating the ionic strength of the assay resulted in less enzyme activity, and reversed haemocyanin conformational changes associated with phosphatidylserine binding (confirmed by fluorescence emission spectra). The addition of wild type and rearranged peptides - mimicking the P181 to K196 region close to the active site of haemocyanin subunit II - to phenoloxidase assays resulted in less product (dopachrome) formation. We propose that non-specific electrostatic interactions between haemocyanin and endogenous activators such as phosphatidylserine facilitate the switch to a phenoloxidase-like enzyme.