Feature Article Comparison of Voltammetry Assisted by Multivariate Analysis with EXAFS as Applied to the Study of Cd- and Zn-Binding of Metallothionein Related Peptides M. S. DÌaz-Cruz, J. M. DÌaz-Cruz, M. Esteban* Departament de QuÌmica AnalÌtica, Facultat de QuÌmica. Universitat de Barcelona, Av. Diagonal 647, E-08028 Barcelona. Spain, e-mail: miquel.esteban@apolo.qui.ub.es Received: July 26, 2001 Final version: October 31, 2001 Abstract Zn- and Cd-complexes of glutathione (GSH) and the metallothionein related peptide Lys-Cys-Thr-Cys-Cys-Ala (FT) were investigated by EXAFS and by voltammetry, assisted by multivariate curve resolution (MCR). EXAFS data from the Zn-FT system seem to show the formation in low extent (under 50%) of a metal cluster with a coordination number of four. In the cases of Cd-FT, Zn-GSH and Cd-GSH no sound conclusions can be deduced from EXAFS. These results are in contrast with those found by voltammetric techniques. The use of MCR allows to calculate the voltammograms corresponding to each single process and its corresponding concentration profile. Then models of complexation are proposed. Keywords: Voltammetry, EXAFS, Multivariate analysis, Glutathione, Metallothionein related peptides, Cadmium, Zinc 1. Introduction Electroanalytical methods offer the possibility to distin- guishamongfreemetalionsandmetalsboundincomplexes of different stability. Mammal metallothioneins (MT) and related peptides as glutathione (GSH) or the Lys-Cys-Thr- Cys-Cys-Ala peptidic fragment (FT), constitutive of the a- domain mouse liver MT I, have been extensively studied by a great variety of polarographic and voltammetric tech- niques. Differential pulse polarography (DPP) is the most extensively used in the characterization of Cd,ZnMT, CdMT and ZnMT from diverse origins [1 ± 6] as well as in Cd-FT, Zn-FT and Cd,ZnFT [7±11] or Cd-GSH and Zn- GSH [12±15]. Other techniques such as square-wave voltammetry (SWV) [16, 17] or cyclic voltammetry (CV) [18 ± 21] have also been applied. In addition to the above mentioned techniques, cathodic (CSV) and anodic (ASV) stripping voltammetry have also been used to characterize the Cd-, Pb-, Zn- and Cu-Phytochelatines metal complex- ation equilibria [22±24]. The use of 113 Cd-NMR and circular dichroism (CD) for the Cd-GSH and Cd-FT systems only confirmed partially the voltammetric findings due to the low solubility of FT complexes and the very small size of GSH [9, 13]. Although all these electroanalytical techniques are not for structural characterization, in some cases tentative hypotheses on the structure of the complexes formed can be formulated from the combination of the information providedbyeitherdifferenttechniquesorexperiments.This hasbeenfacilitatedbytherecentuseofmultivariateanalysis techniques such as multivariate curve resolution with alternating least squares optimization (MCR-ALS) [9 ± 15, 17, 20, 21]. X-ray crystallography is the most powerful structural technique, however some proteins refuse to crystallize. Thus, alternative structural determinations are needed. The availability of synchrotron radiation has allowed the development of EXAFS (extended X-ray absorption fine structure spectroscopy), which is ideally suited to probe the immediate environment of specific atoms in biological, chemical and physical systems, irrespective of its state [25]. For a metalloprotein, EXAFS is capable of providing information on the number, type and distance of ligand atomsfromametalatom[26±29].Inaddition,EXAFSdoes not require crystalline material; thus, aqueous protein samples are probed under a variety of conditions. Addi- tional information on the EXAFS theoretical basis and application can be found elsewhere [30 ± 33]. An area where EXAFS can make a substantial contribu- tion is in the study of the zinc environment in metal- loproteins. As zinc is ™silent∫ to most traditional spectros- copies, the characterization of Zn sites is extremely difficult in the absence of a crystalline structure. Abrahams et al. published the earliest EXAFS studies on MT of pig and rabbit throughout the study of the Cd [26], Cu and Zn K- edge EXAFS [27] at 77 K, providing the first information concerning the internal arrangement of zinc atoms in Zn 7 - MT. Later, other works have been accomplished, providing quite similar metal-metal and metal-sulfur distances [28 ± 30]. The Cd-GSH system has been also studied by EXAFS [29],butweassumethatnoconclusiveresultswereobtained becausenodetailsabouttheresultsarereported.Tothebest 899 Electroanalysis 2002, 14, No. 13 ¹WILEY-VCH Verlag GmbH, 69469 Weinheim, Germany, 2002 1040-0397/02/1307-0899 $ 17.50+.50/0