Hemocyanin of Octopus vulgaris. The molecular weight of the minimal functional subunit in 3 M urea

Abstract

In the presence of 3 M urea Octopus vulgaris (Mollusca) hemocyanin dissociates completely, giving a single functional component which reassociates in the original aggregate after removal of urea. The molecular weight of this subunit has been determined by gel filtration, by viscosity measurements, and by ultracentrifugation. The values obtrained with the different methods range from 247,000 to 262,000. Electron microscopy shows that the reassociation of the functional subunit after removal of urea is complete and gives the typical cylindrical structure of the native protein. This component is the minimal functional subunit which can be obtained from Octopus hemocyanin without splitting covalent bonds. It is suggested that this component is made by five protomers (50,000 daltons) containing one oxygen binding site each bound covalently through, perhaps, the carbohydrate moieties of the protein.

Hemocyanin of the Molluscan Concholepas concholepas Exhibits an Unusual Heterodecameric Array of Subunits

“…removal of divalent cations (11)(12)(13)(14), pH changes, and the addition of denaturing agents (15,16)), have helped to elucidate its subunit composition and structure. In turn, the uses of immunochemical methods combined with partial proteolytic digestion of the subunits, supported by sequence studies, have allowed researchers to establish structural relationships among different FUs and their location in the polypeptide chain (17)(18)(19)(20)(21)(22).…”

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

“…Numerous methods have been developed along the decades to obtain information about mass, size, composition, subunit arrangements and physical or biochemical constants . Commonly used methods in this field are sedimentation velocity (SV), sedimentation equilibrium (SE), gel electrophoresis, size‐exclusion chromatography (SEC), STEM, cryoelectron microscopy (cryo‐EM), crystallography, primary sequence determination and nuclear magnetic resonance . All the cited techniques are of major interest for investigation of protein mass and structure: they can either provide high quality information (SE, SV, STEM, crystallography and cryo‐EM) or provide rapid and convenient analysis (gel electrophoresis, SEC).…”

“…Due to the high accuracy of molecular mass determination of the mass spectrometric techniques, they also allow determination of molecular masses of FUs and post‐translational modifications, such as glycosylation . Until now, several FUs were isolated and analysed after enzymatic cleavage of Hcs . The molecular masses of the fractions obtained after tryptic digestion of Rapana with trypsin and separated by a FPLC system using a Q sepharose high‐performance column were measured by MALDI‐TOF‐MS (Figure ).…”

“…[30] Commonly used methods in this field are sedimentation velocity (SV), sedimentation equilibrium (SE), gel electrophoresis, sizeexclusion chromatography (SEC), STEM, cryoelectron microscopy (cryo-EM), crystallography, primary sequence determination and nuclear magnetic resonance. [8,11,23,[30][31][32][33][34] All the cited techniques are of major interest for investigation of protein mass and structure: they can either provide high quality information (SE, SV, STEM, crystallography and cryo-EM) or provide rapid and convenient analysis (gel electrophoresis, SEC). These techniques were applied to determine the molecular masses of several molluscan and arthropodan Hcs.…”

“…[38][39][40] Until now, several FUs were isolated and analysed after enzymatic cleavage of Hcs. [14,23,31,41,42] The molecular masses of the fractions obtained after tryptic digestion of Rapana with trypsin and separated by a FPLC system using a Q sepharose high-performance column were measured by MALDI-TOF-MS ( Figure 6). The ions at m/z 50 585, 55 978 and 59 123 are due to three FUs, while the ions at m/z 97 431 and 108 150 are due to two linked FUs.…”