Developmental changes in the structure and function of lobster hemocyanin
Olson, Kirby S.
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Respiratory systems function as one of the interfaces between an organism and its environment; the flexibility of this system may therefore constrain the distributional limits of the organism. The respiratory system of a substantial number of marine invertebrate species, particularly amongst the decapod crustaceans, contain hemocyanin as the oxygen binding protein. Previous investigations have revealed a great deal about the structural and functional properties of this respiratory pigment, but a number of important questions remain unanswered. No strong correlation has yet been established between differences in the structure of the oligomer or the diversity of its subunits and the functional properties of the protein. The link between anatomy, habitat, and hemocyanin structure or function is also still unclear in many areas. This thesis was designed to examine aggregate size, subunit composition, and oxygen binding properties of hemocyanin in the larval, juvenile, and adult stages of Homarus americanus. These studies elucidate the relationship between structure and function of hemocyanin, and provide some insights on larval ecology as well. Hemocyanin occurs in all larval stages of the American lobster in concentrations between 8 and 12 mg/ml; concentration in the adult is about five times that of the larval stages. Calculations of cardiac output based on preliminary measurements of heart rate and heart size for the first three larval stages indicate that the oxygen bound to the hemocyanin may be required for routine respiration. The ratio of hexameric and dodecameric forms of hemocyanin were determined for each of the stages under study. Stage I and II larvae possess almost exclusively hexameric hemocyanin, but stage III larvae have almost equal proportions of the two forms. The dodecamer predominates in the fourth stage larvae, and is the only form found in the juvenile lobster. Adult lobster hemolymph appears to contain both forms, but only the dodecameric hemocyanin has an absorption peak characteristic of oxygen binding at the hemocyanin active site. Oxygen binding curves were constructed for all stages and showed sigmoid stages I, II, and IV and the juvenile were similar to each other and showed no significant change between the two temperatures. Curves for stage III had a position and shape intermediate to that of the other larval stages and that of the adult. The Bohr shift showed a significant increase at stage III, but no additional significant changes occurred in the slope prior to or subsequent to that stage. Cooperativity showed no trend with pH at any stage, except possibly in stage I. SDS-PAGE gels of the hexamer and dodecamer of hemocyanin in each of the stages of the lobster provided information on the number of types of monomers in the aggregates. The larvae starts out with a hexameric hemocyanin consisting of a single type of monomer. At the third stage a second type of monomer is added. This type of monomer is present in small amounts in the stage III hexamer, but in the dodecamer of stage III it occurs in the same proportion as the first type of monomer. The presence of the second type of monomer in an equal concentration correlates with the appearance of a substantial proportion of dodecameric hemocyanin. These two structural changes are reflected in a significant increase in the slope of the Bohr shift. In adult lobsters a third type of monomer is present; there is no change in aggregate size or Bohr shift at this point, but the oxygen tension requiresd for saturation of half the sites on tyhe pigment (the P50) is significantly lower. Addition of SDS types of monomer occurs during larval development in the lobster and appears to alter aggregate size, response to pH changes, and the P50 although cooperativity is unaffected.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 1991
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