Plasma and Hemocyanin Phenoloxidase Derived from the Hemolymph of Giant Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879)

Abstract

We attempted to study the immune response in M. rosenbergii by melanization reaction produced by plasma phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates, and it was found that the diphenols were only oxidized. The enzyme was characterized as catechol oxidase type of PO and L-3,4 dihydroxyphenylalanine (L-DOPA) showed the highest substrate affinity to the enzyme. The biochemical parameters that determined optimum enzyme activity were found to be 2.5 mM L-DOPA at an absorbance of 470 nm, 10 mM Tris–HCl buffer at pH 7.5, temperature at 25°C, and 15 min incubation. Kinetic characteristics of plasma were studied from the M. rosenbergii. The hemocyanin was isolated by gel filtration chromatographic technique using Sephadex G-100. The M. rosenbergii hemocyanin (MrHC) showed only one band with a molecular weight of 325 kDa on native polyacrylamide gel electrophoresis (PAGE) when stained with Coomassie Brilliant Blue (CBB) and bathocuproine sulfonic acid. The reduction of MrHC protein in SDS-PAGE displayed three subunits with a molecular weight of 74, 76, and 78 kDa, respectively. Determination of optimal condition for PO activity of plasma has also been attempted. The plasma optimal condition taken for the MrHC was tested for its ability to oxidize diphenols such as L-DOPA was shown only PO activity. These results showed that in the presence of PO and peroxidase inhibitors, phenylthiourea (PTU) and tropolone respectively have decreased plasma and MrHC PO activity. This indicates that hemocyanin triggers innate immunity probably through one of its subunits that function as the active moiety.