ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences.