Fluorescence properties and stability of dioxygen--binding functional units from the Rapana thomasiana hemocyanin subunit RHSS2.

Molecular aggregates of the Rapana thomasiana hemocyanin are composed of two structural subunits, RHSS1 and RHSS2, each of which contains eight functional units (FUs) reversibly binding dioxygen. Multiunit fragments and individual 50-60 kDa FUs from RHSS2 were isolated and characterized by electron and fluorescence spectroscopy. The units have similar fluorescence parameters demonstrating that the tryptophyl side chains are located in the hydrophobic core of the globular folded regions. The copper-dioxygen system at the binuclear active site stabilizes considerably the native protein structure and quenches the indole emission. The removal of this system decreased the 'melting points' drastically Tm by 13-20 degrees C and increased 2-4 times the fluorescence quantum yields. The individual FUs differ considerably in their thermostability. The activation energy for the thermal deactivation of the excited tryptophyl residues of the apo-FUs is lower compared to that of the whole molluscan apo-Hcs.