Interaction of uranium(VI) and europium(III) with alpha-amylase

The calcium-binding protein α-amylase (α-1,4 glucan-4-glucanhydrolase) catalyzes the degradation of starch and glycogen. It is widely distributed in plants, animals, and microorganisms. In saliva and pancreas it is a main enzyme which is built in high amounts. Incorporated heavy metals like actinides firstly come into contact with saliva. In order to explore the origin of the well known toxic effect of these metals we investigate the interaction between UO22+ and Eu3+ (as an inactive analogue for threefold actinides) with the main constituents of saliva like α-amylase.

The influence of UO22+ and Eu3+ on the enzyme activity was tested with the α-amylase assay method by Bernfeld [1]. Both metal ions have a strong inhibition effect even at very low metal to α-amylase ratios. Sorption experiments showed a high binding capacity of α-amylase and that in the sorption maximum (pH 6-7) the protein can bind up to a tenfold molar excess of the metal ions. Both metal ions displace the Ca2+ ion, which is the central metal ion in the protein, even at equimolar amounts. UO22+ and Eu3+ seem to occupy first the Ca2+ binding place and then other potential metal binding sites in the protein. The binding process occurs in a short time, so that after 5 minutes the main metal amount is accumulated by α-amylase.

The UO22+ and Eu3+ α-amylase complexes were investigated using time-resolved laser-induced fluorescence spectroscopy (TRLFS) and extended x-ray absorption fine structure (EXAFS) spectroscopy. In case of UO22+ and TRLFS, we observed in the pH range 3-7 a red shift of the peak maxima and two different luminescence lifetimes, indicating the formation of two different UO22+ protein complex types. With EXAFS spectroscopy two different binding types were identified. At equimolar metal to ligand ratio we identified a fourfold coordination of UO22+ with only monodentate binding carbonyl groups. With excess of UO22+ the binding properties changes and higher coordination numbers are observed with indication of monodentate and bidentate binding carbonyl and carboxylate groups. For the Eu3+ α-amylase complex, the luminescence lifetimes show that 2 or 3 water molecules remain in the first coordination sphere. EXAFS measurements points at the presence of monodentate and bidentate coordinated carbonyl or carboxylate groups.

[1] P. Bernfeld, Methods in Enzymology 1(1955) S. 149-158