Structure–function relationships in glutathione and its analogues Artur Kre ˛z ˙el a and Wojciech Bal * b a Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland. E-mail: arti@wcheto.chem.uni.wroc.pl; Fax: +48 71 3282348; Tel: +48 71 3757264 b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin ´skiego 5a, 02-106 Warsaw, Poland. E-mail: wbal@ibb.waw.pl; Fax: +48 22 6584636; Tel: +48 22 6597072 ext. 2353 Received 8th August 2003, Accepted 17th September 2003 First published as an Advance Article on the web 6th October 2003 The results are presented of measurements of protonation constants (potentiometry and NMR), UV spectroscopic properties and redox potentials of GSH and its five analogues, which are modified at the C-terminal glycine residue (γGlu–Cys-X, X = Gly, Gly–NH 2 , Gly–OEt, Ala, Glu, Ser). Strong linear correlations were found between various properties of the thiol and other functions of these peptides. These results allow discussion of the relationships between the structures and properties in glutathione and its analogues, and provide a novel chemical background for the issue of control of GSH reactivity. Introduction Glutathione (GSH, γGlu–Cys–Gly) is one of the most ubi- quitous and important small biomolecules, present in cells of all organisms at millimolar concentrations, and possessing a multitude of physiological functions. 1 It is also one of the most widely studied molecules, with more than 120000 references in the literature. 2 The major, well established functions of glutathione include redox-buffering of the cell environment, detoxication of xenobiotics (including drug resistance in cancer cells) and antioxidant activity, including the maintenance of biological membranes. Novel roles of glutathione are being recognized continuously. The recent additions include the release of zinc from metallothionein (a specific function for glutathione disulfide, GSSG), 3 protein glutathionylation as an element of intracellular signal transduction, 4 and intracellular nitric oxide storage and transport. 5 Glutathione is also a versatile chelator of many different metal ions, with con- sequences in toxicology and homeostasis. 6,7 Virtually all of these functions depend on the reactivity of the thiol group of glutathione. The presence of a γ-peptidic bond between Glu and Cys residues is the most distinct structural feature of glutathione. It is thought to protect GSH from intracellular aminopeptidases. 1 It has two structural consequences: it separates the thiol from the functional groups of the Glu residue and it yields an α- amino acid-like domain at the Glu residue, which is absent from α-peptides. One well established consequence of these features is the above-mentioned versatility of metal ion binding. 6 Another may be the high conformational flexibility of GSH, important for its interactions with enzymes. 8 Many studies were devoted in the past to the acid–base properties of GSH, mainly as a prerequisite for metal binding experiments. The IUPAC Stability Constants Database lists 19 references, published between 1953 and 1994, 9 with newer ones coming up at a pace of ca. one a year. Those relevant for our results are referred to in the Discussion section. The redox potential of the 2GSH–GSSG pair was estab- lished accurately, with various methods 10,11 and was discussed recently in depth in the context of cellular physiology. 12 We have re-established acid–base and redox properties of GSH and compared them with those of its several analogues, which were modified at the C-terminus by substituting the Gly carboxyl function, or replacing Gly with another amino acid residue. These analogues are presented in Scheme 1. Such com- parative studies were not performed previously and several of these derivatives are novel. Our approach provided a new look at the properties of GSH, in particular by delivering evidence for the conformational preferences present in the molecule, which control its acid–base and redox properties. Materials and methods Materials Glutathione (GSH), glutathione glycine ethyl ester (γGlu–Cys– Gly–OEt, γECGOEt), glutamic acid (Glu), sodium (3-tri- methylsilyl)-2,2,3,3-tetradeuteriopropionate (TSP), 5,5'-dithio- bis-2-nitrobenzoic acid (DTNB), sodium phosphates and NaOD (40% w/v in D 2 O) were obtained from Sigma. KNO 3 , HNO 3 , NaClO 4 , and NaOH volumetric solution (0.1 M) were purchased from Merck. D 2 O (99.9%) and DCl (35% solution in D 2 O) were from Cambridge Isotope Laboratories. Peptide synthesis The peptides γGlu–Cys–Ala, γGlu–Cys–Ser, and γGlu–Cys– Glu were synthesized in the solid state on a 2-chlorotrityl chloride resin, while γGlu–Cys–Gly-am was synthesized using the H-linker-2-chlorotrityl resin. Fmoc strategy was used. 13,14 The N-Fmoc-protected amino acids N–Fmoc–Gly–OH, N–Fmoc–Ala–OH, N–Fmoc–Ser(tBu)–OH, N–Fmoc–Glu- (γ-tBu)–OH and N–Fmoc–Cys–(Mtt)–OH were obtained from Scheme 1 Structures of γGlu–Cys–Xaa peptides studied in this work, presented in their fully protonated forms. DOI: 10.1039/ b309306a 3885 This journal is © The Royal Society of Chemistry 2003 Org. Biomol. Chem. , 2003, 1, 3885–3890 Published on 06 October 2003. Downloaded by Duke University on 14/07/2014 20:23:37. View Article Online / Journal Homepage / Table of Contents for this issue