SUBUNIT HETEROGENEITY IN ARTHROPOD HEMOCYANINS .2. CRUSTACEA

The hemocyanins of 10 decapod Crustacea were dissociated and their subunits analyzed by high resolution polyacrylamide electrophoresis (PAGE): 5 brachyuran crabs (Cancer pagurus, Carcinus maenas, Macropipus holsatus, Hyas araneus, Maja squinado), 3 Astacura (Astacus leptodactylus, Homarus americanus, Homarus gammarus) and the spiny lobsters Palinurus vulgaris and Panulirus interruptus All of the species save the spiny lobsters possess a major hemocyanin component sedimenting with 24 S. A second hemocyanin component sedimenting with ca 16 S was found in H. gammarus, M. squinado, C. pagurus and M. holsatus (about 10 per cent in each case) and in A. leptodactylus (about 25 per cent). A second, major blood protein (10–25% of the total blood protein) was observed in H. gammarus where its sedimentation coefficient was 24 S, M. squinado (16 S), H. araneus (24 S) and C. pagurus (16 S). This second protein has no respiratory function. Two such non-respiratory proteins sedimenting with 24 S and 16 S were found in H. americanus. Between 2 and 7 hemocyanin bands were obtained after incubation with sodium dodecylsulfate (SDS) and β-mercaptoethanol and subsequent electrophoresis in polyacrylamide gradients. The average molecular weight was about 75,000 in the crabs, 80,000 in the crayfishes and 85,000 in the spiny lobsters. The non-respiratory proteins yield between one and four chains with molecular weights ranging from 76,000 to 87,000. The hemocyanins were dissociated at pH 9.6 into “native” subunits, but dissociation was not quantitative in several species. By gel filtration, the products were separated into undissociated material and hemocyanin monomers (5 S). In Astacus leptodactylus a dimeric subunit (7 S) was obtained in addition; its components are linked by a disulfide bridge. The subunit mixtures were separated by PAGE into 4 to 6 distinct bands. To establish the total number of different polypeptide chains present in each hemocyanin, the two electrophoretic patterns were related to each other by preparative isolation of “native” subunits and subsequent analysis in SDS-PAGE. The number of different polypeptide chains ranges from four to seven in the species studied by us. In those species which contained both 24 S and 16 S hemocyanin, more different polypeptide chains were found in the 24 S hemocyanin than in the 16 S hemocyanin, the only exception being Homarus gammarus. © 1979, Springer-Verlag. All rights reserved.