Phenoloxidase in the periostracum of the marine bivalve Modiolus demissus dillwyn

Phenoloxidase was extracted from Modiolus demissus mantles and periostraca with the detergent sodium dodecyl sulfate. Partial purification of the periostracal enzyme was achieved by gel filtration and polyacrylamide gel electrophoresis. The enzyme, present in the mantle in latent or proenzyme form, can be chymotryptically activated. Molecular weights of the proenzyme and active enzyme as determined by SDS gel electrophoresis were 80,000 and 70,000 daltons, respectively. A considerable portion of phenoloxidase activity in the periostracum is associated with a large macromolecule that cannot be reduced into smaller subunits, suggesting enzyme covalently immobilized by quinones. Optimal pH of 8.0–8.5 was found for the active enzyme (MW 70,000). Activity was diminished by adding copper chelators or by reducing disulfide bonds. In the former case, activity was restored by titrating the inhibitor with divalent copper. A hydrophobic active site on the enzyme is suggested by low Km and high reaction velocities for substrates with high partition coefficients in 2‐octanol:water such as 4‐methylcatechol and 4‐butylcatechol.