Positions of the glycans in molluscan hemocyanin, determined by fluorescence spectroscopy.

Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) and also their structural subunits (RvH1, RvH2 and βcHlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the didecameric form of the molecules. Titration with the structural subunits βcHlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural subunit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of βcHlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.