Reference: Biol. Bull. 171: 90-1 15. (August, 1986) EVOLUTION AND FUNCTION OF STRUCTURALLY DIVERSE SUBUN1TS IN THE RESPIRATORY PROTEIN HEMOCYANIN FROM ARTHROPODS JURGEN MARKL Zoologisches Institut, Universitdt Miinchen, Luisenstr. 14, D-8000 Miinchen 2, Federal Republic of Germany ABSTRACT Native aggregation level and subunit composition of the hemocyanins from 86 adult chelicerates and crustaceans, and from the larval stages of 2 crabs, were analyzed by means of electron microscopy, polyacrylamide electrophoresis, immuno blotting, and crossed immunoelectrophoresis, supported by a variety of preparative separation techniques. The up to eight immunologically discernible subunit types were interspe-cifically correlated, classified, and evolution lines derived. Phylogenetic consequences are discussed, and are particularly aggravating in spiders. A single subunit suffices for the formation of hexamers (1 X 6). In the architecture of higher-ordered hemocyanins, the various subunits act as building-blocks of distinct specification. This was studied in 2 X 6 molecules from a hunting spider and several crustaceans, and in 4 X 6 hemocyanin from a tarantula. The various subunits are present in constant proportions. The total set is required to reorganize the original aggregate from subunit mixtures. Stable oligomeric segments of native hemocyanin particles revealed the gross distribution of the diverse subunits. Immuno electron microscopy of the native hemocyanins decorated with monospecific Fab fragments showed the exact topographic position of each subunit type, and detailed models of the quaternary structure could be derived. The oxygen binding function of 4 X 6 hemocyanin from the tarantula Eurypelma californicum is excessively modulated by subunit interaction phenomena. We measured native, reassembled, and mercury-blocked 4 X 6-mers, oligomeric segments, single subunits, and reassembled 4 X 6-mers with one subunit type chemically modified. The spatial range of allosteric interaction, and specific contributions of the diverse subunits are outlined. INTRODUCTION Hemocyanin, a blue copper-protein, functions as an oxygen carrier in the blood of arachnids, horseshoe crabs, crustaceans, and centipedes. Another hemocyanin occurs in inkfishes, chitons, many snails, and some primitive bivalves; however, despite certain similarities, molluscan hemocyanin differs decisively from arthropod hemocyanin in its main structural features, and therefore will not be discussed here. Most certainly, both pigments have evolved independently from tyrosinase, an ancestral precursor. Arthropod hemocyanins are multi-subunit proteins with a molecular mass of about 75,000 per polypeptide chain. The polypeptides or subunits are arranged as cubic hexamers (1 : 6), or multiples of hexamers (2 X 6, 4 X 6, 6 X 6, 8 X 6); the native aggregation level is species-specific. (Ghiretti, 1968; Van Holde and van Bruggen, 1971; Van Holde and Miller, 1982; van Bruggen et at, 1982; Ellerton el at, 1983; Received 4 February 1986; accepted 29 May 1986. Present address: Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Hei-delberg, Federal Republic of Germany. 90
BioStor
