Studies on biochemical characteristics of portunus trituberculatus hemocyanin using immunological methods

Abstract

Hemocyanin of Portunus trituberculatus was purified. SDS polyacrylamide gel electrophoresis (PAGE) were performed to investigate size homogeneity of hemocyanin. The electrophoretic pattern of purified hemocyanin with detergent, SDS in 7.5\% gel revealed a single band with a molecular weight of about 75,000 indicating a size homogeneity of the Portunus trituberculatus hemocyanin subunit. The purified hemocyanin was then subjected to alkaline electrophoresis without detergent. DAVIS-PAGE of hemocyanin in pH 7.5 showed polymorphism of monomers, hexamers, and higher polymers. In order to determine the charge heterogeneity of monomeric subunits, DAVIS-PAGE were performed. While SDS-PAGE showed no size heterogeneity of the monomeric subunit of Portunus trituberculatus, DAVIS-PAGE in high acrylamide concentration showed at least four monomeric subunits. In order to confirm the charge heterogeneity of Portunus trituberculatus hemocyanin, continuous polyacrylamide gel electrophoresis was performed. While nondissociating buffer (Tris/HCl-$CaCl_2$ buffer pH 7.5) demonstrated only polymeric forms without monomeric fractions, continuousPAGE in a dissociating buffer (Tris/HCl_EDTA buffer pH 8.9) showed that monomeric subunits of Portunus trituberculatus hemocyanin were resolved into four distinct bands, designated as I, II, III, and IV. This agreed with the results of DAVIS-PAGE in a nondissociating buffer to observe and isolate the polymeric forms of hemocyanin, and in a dissociating buffer to obtain the monomeric subunits of hemocyanin. To investigate the antigenic heterogeneity of hemocyanin subunits, the experiments of immunoelectrophoresis using anti-whole Portunus trituberculatus hemocyanin antiserum were carried out. The crossed immunoelectrophoresis (CIE) of whole hemocyanin revealed four distinct peaks, as four monomeric bands were observed in continuous-PAGE. These four peaks did not show any partial identity with each other, which suggested the occurrence of at le...