Conversion of phenoloxidase and peroxidase indicators in individual haemocytes of Mytilus edulis specimens and isolation of phenoloxidase from haemocyte extract

Abstract

Haemocytes oxidized 3-amino-9-ethylcarbazole and other peroxidase indicators such as 3,3′-diaminobenzidine·4HCl, 3,3′,5,5′ tetramethylbenzidine·2HCl and 4-chloro-1-naphthol without addition of H₂O₂ indicating that the reaction was possibly not caused by a peroxidase. As these chromogens were also converted by a mushroom phenoloxidase in the absence of H₂O₂, cell smears were incubated with known substrates of phenoloxidases. One of these, l-dopa, caused strong melanin formation in several haemocytes and the reaction could be blocked by a variety of inhibitors including KCN, NaF, 1-phenyl-2-thiourea, cysteine, glutathione, ascorbic acid and HgCl₂. The enzymatic activity was isolated using a concanavalin A column and separated into two fractions with an ion-exchange cartridge. The molecular weights of the glycoproteins were estimated to be 381±13.7 kDa and 316±11.1 kDa. After isoelectric focusing of a haemocyte extract and the two ion-exchange peaks, seven enzyme bands were detected with isoelectric points between pH 5.0 and 5.5. The isolated enzyme fractions both converted 3,3′,5,5′-tetramethylbenzidine·2HCl best at pH 5–6 and l-dopa at pH 7.0 without addition of H₂O₂. Heat-treated cells lost their enzymatic activity; however, a group of haemocytes still bound preoxidized 3-amino-9-ethylcarbazole (= AECox). Also, some of the phenoloxidase inhibitors mentioned above blocked this non-enzymatic staining reaction. About 30–57% of haemocytes from individual mussels were AECox-positive, whereas Mytilus specimens without phenoloxidase-containing cells often occurred. Haemocytes containing this enzyme exhibited a high mobility and a large percentage of them belonged to a cytotoxic cell population.