Interaction of Uranium(VI) towards glutathione – an example to study different functional groups in one molecule.

Glutathione is a ubiquitous compound in living systems. It is a tripeptide containing besides two carboxylic groups also a thiol group as well as amino groups. Glutathione has antioxidant properties and therefore it helps to protect cells against reactive oxygen species. In plant cells glutathione is essential for the stress management. Due to its carboxyl groups and the thiol group the peptide may contribute to the metal complexation.
We have studied the complex behavior towards uranium(VI) exploiting several spectroscopic techniques, like UV-Vis and fluorescence measurements /1/. Direct UV-Vis measurements of the absorption spectra of the uranylspecies lead to a stability constant of log β121 = 38.70 ± 0.15. The glutathione itself does not absorb any light in the spectral range from 350 nm to 500 nm. Additionally the glutathione can be modified by fluoropyrovate in order to generate a species absorbing light in the wavelength range around 300 nm. The pyruvate group substitutes the proton of the thiol group. From these measurements a stability constant for the uranyl-glutathione complex was assigned to be log β121 = 38.85 ± 0.08.
Secondly we exploited the fluorescence properties of the uranyl ion. At a pH of 7.4 the main uranium species in carbonate free solutions are the hydroxospecies (UO2)3(OH)5+ and (UO2)4(OH)7+. By increasing the concentration of glutathione the fluorescence of these species disappears as consequence of the formation of a uranyl-glutathione complex. The stability constant using the Stern-Volmer equation was derived to be log β121 = 38.65 ± 0.02.
Glutathione itself doesn’t show any florescence properties. However, it is known, that the derivatisation of the thiol group by monobrombimane leads to a fluorescent glutathione species. In analogy to the measurements of the fluorescence of the uranylspecies the fluorescence of this conjugate also disappears upon complex formation with heavy metal ions. The resulting stability constant for the uranyl complex was derived to be log β121 = 38.96 ± 0.02 neglecting that the thiol group was modified.
Summarizing all studies the stability constant for formation of a 1:1 uranyl-glutathione complex can be assigned to be log β121 = 38.79 ± 0.15.
As the stability constants the measurements of the pure and the modified glutathione agree very well, we can conclude that the thiol group is not involved in the complex formation. Additionally it could be shown that glutathione does not reduce the uranium(VI) under the experimental conditions.
Comparing these data with stability constants for several flavonoids, showing also very high complex stability constants towards uranium, we conclude that glutathione in plant cells is much more involved in heavy metal stress reactions than flavonoids.

/1/ Diploma thesis L. Frost, TU Dresden, 2009