Hemocianina de megalobulimulus ovatus : aspectos estruturais e funcionais

AUTOR(ES)
DATA DE PUBLICAÇÃO

1983

RESUMO

The hemocyanin from Megalobulimulus ovatus, gastropod of terrestrial habitat, was purified. It was shown the presence of at least two components, c* hemocyanin (71.5% of total hemocyanin), which dissociates in 1 M NaC 1 solution and 3 hemocyanin (28.5% of total hemocyanin) which remains intact in the same conditions. B hemocyanin was isolated by preparative ultra-centrifugation in 1M sucrose solution containing 1 M N a C1 , and some of its functional and structural properties were analysed. This hemocyanin was a glicoprotein containing about 1% in carbohidrates, showed only one sulphydryl group per functional subunit and this number remains after the copper removal. Electron microscopy revealed a pattern quite similar to the hemocyanins from other gastropods, presenting cylindrical molecules with a diameter of 30 nm and 35 nm high. The stability region of the undissociated molecule was determined, and the influence of Ca2+ and H+ ions on the protein dissociation was studied. Four different species were found; 109 S, corresponding to the undissociated molecule; 65.5 S, corresponding to dissociation into halves; 22 S, corresponding to 1/10 molecules, and 13 S, the last degree of dissociation achieved, corresponding to 1/20 of the original molecule. The presence of 20 mfl Ca2+ prevents the dissociation beyond the 65.5 S species up to pH 9.5. Antisera against the whole (109 S) and the dissociated (13 S) species were obtained, double immunodiffusion tests were performed, and no antigenic difference could be observed. The apo-hemocyanin, however, showed only partial identity with the holo-protein, suggesting a possible structural role played by the copper atoms. The presence of oxygen in the binding site showed a protection effect on copper removal by cyanide, at the beggining of the reaction, when comparing oxy and deoxy-hemocyanins. The total hemolymph presented P 5q = 8 .2 mmHg and n^Q= 2.1 at pH 8.1. The undissociated and dissociated purified 6 hemocyanin, at this same pH, presented P50=7,02 mmHg, n50= 1.7 and P5Q- 2.80 mmHg, n5Q = 0.9 respectively. Throughout the studied pH, the dissociated hemocyanin showed to be non cooperative (n^q= T) and its oxygen affinity was always higher than the whole hemocyanin. This showed a reverse Bohr effect of 0.18, six times greater than that found for the dissociated molecule in the pH region 7.5 to 8.5.

ASSUNTO(S)

proteinas molusco - fisiologia

ACESSO AO ARTIGO

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