Biochemical and Biophysical Investigations on the Hemocyanin Subunits of Ovalipes Catharus and Jasus Edwardsii

Abstract

Amino acid analyses on Jasus edwardsii and Ovalipes catharus hemocyanins indicated that these proteins were typical of most Arthropod hemocyanins in that they contained large amounts (23%) of aspartic and glutamic acids. Sedimentation velocity studies on Ovalipes hemocyanin revealed that this protein dissociated to give subunits with S20,w values of 25.5s 17.1s and 5.4S. This hemocyanin was stabilized against dissociation up to PH 10.6 by Cac12. Sedimentation equilibrium studies gave molecular weights of 888,200 for the 25S subunit and 76,600 for the 5S subunit. The subunits of both hemocyanins were analysed at PH 7.8, 8.8 and 10.6 by PAGE, on varying gel concentrations, and Ferguson plots prepare Five PAGE bands were obtained for both hemocyanins at PH 7.8. These protein bands represented two 25S charge isomers, two 16S charge isomers and a third component, a size isomer (MW 1.30-1.36x106). Similar plots at PH 8.8 revealed a total of 13-14 protein bands for both hemocyanins. These proteins could be grouped into a series of charge and size isomers ranging in molecular weight from 70,000 to 1,380,000 depending upon the species. At pH 10.6, the 5S hemocyanins produced three bands on electrophoresis. Molecular weights obtained for these 5S bands were, for Jasus edwardsii, 86,500 (72%), 97,000 (10%) and 117,000 (18%) and for Ovalipes catharus, 87,500 (89%), 98,500 (5%) and 113,000(6%). Larger protein, 9-l0S (MW 290,000) gave ca. 3 extra bands. SDS-DTT-PAGE studies on the native hemocyanins gave three monomers with molecular weights for Jasus edwardsii of 64,000 (12%), 98,000 (13%) and 110,000 (75%), and for Ovalipes catharus, 69,000 (9%), 101,000 (27%) and 112,000 (64%). A theory is advanced on the basis of PAGE, and other analyses, which attempts to solve the difficulty of assigning the 75,000 molecular weight unit as the fundamental monomer for arthropod hemocyanins. Reassociation experiments on the hemocyanins indicated that it was possible to get 85% association to the 16S and 25S particles at pH 7.0 after prior dissociation at pH 8.8. For samples dissociated at pH 10.6, reassociation to 25S particles occurred at pH 7.0 only in the presence of CaCl2 for Ovalipes, but the 16S hexamer was the largest particle observed under these conditions for Jasus. Column chromatography separated an association-dissociation equilibrium of 9-10S and 5S particles at pH 10.6. The purified 9-l0S particles contained copper' but the protein did not possess an A338' whereas the 5S component contained larger quantities of copper and, the protein exhibited an A338. The copper on both subunits was removed by extended dialysis at pH 10.6 in glycine-EDTA buffers. Removal of the copper resulted in a concentration shift in favor of the 9-10S particle accompanied by a breakdown of the 5S particles to give a copper-enriched protein. Isoelectrofocusing of both hemocyanins showed that the native proteins were comprised of 5 components with pI of ca. 4.6-4.8. Ion exchange of both hemocyanins in Tris-glycine-EDTA buffers at pH 8.8 separated the charged components into two fractions which were heterogeneous by PAGE and SDS-PAGE analyses. Extended dialysis of hemocyanins in the ion exchange buffer led to a breakdown in some of the protein to give a component, with an A255 maximum, containing little copper. A bathochromic shift was observed in the far-ultraviolet absorption spectra of hemocyanins treated with EDTA. Further investigations revealed that a perturbation phenomenon was taking place which may have involved weak binding of BDTA to the aromatic side chains and cystine, especially predominant for tryptophan. The bathochromic shift induced was entirely reversible upon titration of the EDTA-treated hemocyanins with CaC12. The effect, of temperature on the far-UV spectra revealed, that both heat denaturation and base- or heat-induced dissociation could result in a decrease in absorbance intensity and a bathochromic shift (base-dissociation only) in the ~210-230 nm region. Oxygen-binding studies have been made at pH 7.0, 8.8 and 10.6 in the presence of CaCl2, or EDTA. The oxygen-binding was cooperative for both species of hemocyanin with calcium and a positive Bohr effect was present (nmaxnn3.0). Removal of all divalent cations with EDTA resulted in a large decrease in cooperativity and the Bohr effect was reduced for Ovalipes hemocyanin at pH 7.0-10.6. For Jasus hemocyanin, the Bohr effect was reversed, at pH 7-8.8 and positive but reduced at pH 8.8-10.6.