EVOLUTION AND FUNCTION OF STRUCTURALLY DIVERSE SUBUNITS IN THE RESPIRATORY PROTEIN HEMOCYANIN FROM ARTHROPODS

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 interspecifically 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 .times. 6). In the architecture of higher-ordered hemocyanins, the various subunits act as building-blocks of distinct specification. This was studied in 2 .times. 6 molecules from a spider and several crustaceans, and in 4 .times. 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 .times. 6 hemocyanin from the tarantula Eurypelma californicum is excessively modulated by subunit interaction phenomena. We measured native, reassembled, and mercury-blocked 4 .times. 6-mers, oligomeric segments, single subunits, and reassembled 4 .times. 6-mers with one subunit type chemically modified. The spatial range of allosteric interaction, and specific contributions of the diverse subunits are outlined.