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Speciation 98: Abstracts
Zoltán Szakács1, Béla Noszál2
1 Eötvös University, Departement
of Inorganic and Analytical Chemistry, H-1117 Budapest, Pázmány s. 2, Hungary;
2 Semmelweis University, Institute of Pharmaceutical
Chemistry, H-1092 Budapest, Hõgyes E. u. 9, Hungary
e-mail: zotyesz@ludens.elte.hu, nosbel@hogyes.sote.hu
Competition often takes place in biological systems between protons and metal ions for coordination to the donor groups of multidentate biomolecules. When groups of similar basicity coexist, their overlapping protonation occurs. At any pH protonation isomers (microspecies) form, which may bear different receptor binding and transport properties. Their distribution can be quantitated by means of protonation micro-constants, which are calculated from the pH-dependence of solution spectra (UV-pH, CD-pH and NMR-pH titrations).
We have systematically examined the protonation sequences of multidentate molecules of various symmetry. The main conclusion is: for more than tridentate molecules all of the microconstants can only be calculated in the most symmetrical (An and An-1B) cases. Due to combinatorial reasons, in less symmetrical situations only some of the microconstants can be obtained unambigously, the calculation of the others demands chemical assumptions (e.g. relative basicities of analogue compounds or independence of the interactivity parameters from the protonation state).
Glutathione (-glutamyl-cysteinyl-glycine), the most abundant biological thiol is the key molecule against oxidative stress in living organisms. The biological activity of GSH has been shown to correlate with the site-specific basicity of its thiolate group, hence the microscopic protonation equilibria of GSH has been thoroughly investigated. However, the GSSG microspeciation is unknown in the most complicated acidic part, when four carboxylate groups protonate simultaneously (A2B2 case).
1H NMR-pH titrations were performed in H2O solutions to characterise the basicities of the individual carboxylate groups. It was proven that not all microspecies concentrations can be evaluated from the experimental data in a purely mathematical way. It was assumed instead, that the basicity-modifying interactions of the carboxylates can be quantified in terms of additive interactivity parameters. Using this model, we were able to calculate all microconstants. The results show that in every stage of protonation, the glycyl carboxylates are by an order of magnitude more basic than the glutamyl ones, as expected. Hence the disulfide formation does not change significantly the relative basicities of the groups. Our studies also quantitate that no specific interaction exists between the four carboxylate sites, rather, the strength of their interactions are similar. To obtain further confirmation, the synthesis and NMR-pH titration of GSSG diethyl ester, a model compound was performed and excellent agreement was found between the corresponding microconstants. The calculations described above allowed the construction of a distribution diagram for all the 10 non identical GSSG microspecies.
