Orientador: Maria Sumiko Arita Matsuura

Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia

Resumo: Hemocianina do Gastropodo anfíbio Ampullaria canaliculata, da região do Pantanal Mato Grossense, mostrou grande similaridade com as hemocianinas de moluscos terrestres e marinhos, especialmente, com relação ao peso molecular e aos tipos de produtos de dissociação. A determinação do peso...

Resumo: Hemocianina do Gastropodo anfíbio Ampullaria canaliculata, da região do Pantanal Mato Grossense, mostrou grande similaridade com as hemocianinas de moluscos terrestres e marinhos, especialmente, com relação ao peso molecular e aos tipos de produtos de dissociação. A determinação do peso molecular da hemocianina de A. canaliculata determinado por cromatografia de exclusão molecular em coluna de Sephacryl S-400 mostrou somente um pico de eluição, tanto na hemolinfa total quanto na hemocianina em presença de Ca¿POT.2+¿ 20 mH e Mg¿POT.2+¿ 20 mH, estimado em torno de 7.5 x 10 'POT.6¿ Da. Hemocianina desionisada de A. canaliculata submetida a filtração em gel resultou na formação de três picos, que através do volume de eluição mostraram ser proteínas de unidade de peso molecular 7.5 x 10¿POT.6¿ Da, 1.0 x 10 Da e 3.2 x 10 5 Da, sugerindo ser espécies moleculares correspondentes a 1/1, 1/10 e 1/20 da proteína nativa. Com a hemolinfa total, recém coletada, foram realizadas experiências de equilíbrio com O2 diretamente, variando apenas os valores de pH, e obteve-se um valor de P50 = 3.5 mm Hg e nH = 2.3 em pH 8.2. O efeito de cátions divalentes sobre o estado de agregação de hemocianina de A. canaliculata, assim como os diferentes picos eluidos da coluna de Sephacryl S-400 são ilustradas por analises imunoeletroforeticas

Abstract: The functional properties of A. Canaliculata hemocyanins, in their ability to transpor oxygen, are very similar to several hemocyanins studie. In this snail the oxygen binding strength increases with increasing pH, exhibiting a normal Bohr shift. The oxygen binding to hemocyanins is also...

Abstract: The functional properties of A. Canaliculata hemocyanins, in their ability to transpor oxygen, are very similar to several hemocyanins studie. In this snail the oxygen binding strength increases with increasing pH, exhibiting a normal Bohr shift. The oxygen binding to hemocyanins is also aífected by presence oí divalent cations. In this context, the two cations Ca¿POT.2+¿ and Mg¿POT.2+¿ act similarly with no changes in the the P and Bohr effect values. The oxygen curve using hemocyanin 50 free of divalent cations showed inconsistent data for P50 association and and nH values, suggesting the production of different dissociation behavior with pH variation. However, the hemolymph showed higher values of log P50 than the hemocyanin in presence of divalent cations. This phenomenum should be explained by the existence of dialyzable factors, not strongly associated withhemocyaninJ which does not affect cooperativity of the oxygen binding and Bohr affect. The A. canaliculata hemocyanin does not exhibit cooperative oxygen binding in the presence of EDTA since on treatment with EDTA to remove divalent cationsJ it dissociates. into several subunits as expected and shown by the immunoelectrophoretic analysis. The molecular weight of A. canaliculata hemocyanin was determined by gel filtration. The native hemolymph, as well as with hemacyanin in the presence of cations Ca¿POT.2+¿ and Mg¿POT.2+¿ showed Mw = 7.5 x 10 'POT.6¿ Da, 1.0 x 10 'POT.6¿ Da and 3.2 x 10 5 Da. However, for the preparation to the same hemocyanin dialyzed of Mw = 7.5 x 10 'POT.6¿ Da were obtained. The fractions eluted from Sephacryl S-400 column as well as effect of Ca¿POT.2+¿ and Mg¿POT.2+¿ on both the stability of hemocyanin and reversibility of products of dissociation caused by EDTA were illustrated by immunologic analysis