The role of the enzyme alpha-amylase in binding of An(III)/Ln(III) by oral ingestion

In case of incorporation, radionuclides represent a serious health risk to humans due to their (radio-)toxicity. Thus, the determination of their speciation and transport on a molecular level is crucial for the understanding of the transport, metabolism, deposition and elimination in the human organisms. In case of oral ingestion of contaminated food or radioactive substances the first contact medium in the mouth is the aqueous biofluid saliva which contains inorganic ions (mainly Na+, K+, Ca2+, Cl-, CO32-, PO43-) and numerous biomolecules, mainly proteins. One of the major proteins in saliva is the digestive enzyme α-amylase which catalyzes the hydrolysis of the α-1,4 glycosidic linkages of polysaccharides like starch or glycogen. [1]
In this study the speciation of curium(III) and europium(III) in saliva as the first contact medium at oral incorporation was investigated with time-resolved laser-induced fluorescence spectroscopy (TRLFS). For TRLFS measurements, fresh saliva samples from human sources have been spiked in vitro with Eu(III) or Cm(III). The identification of the dominant species was achieved by a comparison of the spectroscopic data with reference spectra obtained from synthetic saliva and the main single components of the biofluid. In the pH range from 6.8 to 7.4 similar spectra were obtained. With respect to reference data, the spectra indicate the formation of a ternary metal complex containing phosphate and carbonate anions and, in addition, a coordination of organic matter, namely α-amylase, to the central metal cation is suggested.
To get more information about the binding behavior of α-amylase various investigations with Eu(III) as inactive analog for An(III) were carried out with porcine pancreatic α-amylase (PPA) which serves as model system for various α-amylase species. Sorption experiments showed a high affinity of Eu(III) to α-amylase in a wide pH range, namely between pH 4 and 8. The analysis of binding isotherms demonstrated that up to 3 Eu3+ ions are bound to one enzyme molecule. Hence, the Eu3+ ions seem to replace the Ca2+ ions, a well-known mechanism in biological systems. The effect of Eu3+ on enzyme activity was determined with the α-amylase assay method by Bernfeld [2]. Eu3+ shows a strong inhibition effect on the enzyme activity, but in the presence of Ca2+ in excess the enzyme activity remains nearly unaffected. This effect might be useful for the refinement of decontamination strategies.

[1] Edgar, W. M., Saliva: its secretion, composition and functions. British Dental Journal 1992, 172, 305-312
[2] P. Bernfeld, Methods in Enzymology 1955, 149-158