J. Mendieta

Cadmium-binding properties of glutathione: A chemometrical analysis of voltammetric data

The complexation of glutathione (GSH) by Cd2+ has been investigated as a model for the coordination chemistry of Cd2+ by thiol-containing peptides. Experimental data obtained by differential pulse polarography (DPP) for different Cd2+-to-GSH concentration ratios at fixed pH have been globally analyzed by the recently proposed multivariate curve resolution (MCR) method. Polarograms from experiments at fixed Cd2+-to-GSH concentration ratios but changing pH values have been univariately deconvoluted at each pH value. In both cases, the obtained single DPP peaks can be associated to specific electrode processes. The results obtained in this work, together with structural data from the literature, allow us to propose a model for the complexation of Cd2+ by GSH involving the formation of Cd(GSH)2 and Cd2(GSH)2 complexes. Moreover, a scheme for the electrode process is also proposed.

Study of the zinc-binding properties of glutathione by differential pulse polarography and multivariate curve resolution

Abstract The complexation between Zn 2+ and glutathione (GSH), in borate buffer, is studied by Differential Pulse Polarography (DPP). Data obtained at pH 8.5 from a titration of Zn 2+ by GSH, i.e. at different GSH-to-Zn 2+ concentration ratios have been analyzed by a recently proposed multivariate curve resolution method. DPP signals obtained within a wide pH range, but at fixed 1.25:1 GSH-to-Zn 2+ concentration ratios, have been resolved individually at each pH value. Results from both sets of experiments can be explained with a complexation model involving the formation of a predominant complex, with a stoichiometry of 2:2 GSHxa0:xa0Zn 2+ .This result is in contrast with those for the Cd 2+ –GSH systems, where two complexes, 2:1 and 2:2 GSHxa0:xa0Cd 2+ , were detected. A scheme for the global electrode process is given.

Multivariate Curve Resolution: A Possible Tool in the Detection of Intermediate Structures in Protein Folding

Different multivariate data analysis techniques based on factor analysis and multivariate curve resolution are shown for the study of biochemical evolutionary processes like conformational changes and protein folding. Several simulated CD spectral data sets describing different hypothetical protein folding pathways are analyzed and discussed in relation to the feasibility of factor analysis techniques to detect and resolve the number of components needed to explain the evolution of the CD spectra corresponding to the process (i.e., to detect the presence of intermediate forms). When more than two components (the native and unordered forms) are needed to explain the evolution of the spectra, an iterative multivariate curve resolution procedure based on an alternating least squares algorithm is proposed to estimate the CD spectrum corresponding to the intermediate form.

Two-Phase Induction of the Nonnative α-Helical Form of β-Lactoglobulin in the Presence of Trifluoroethanol

Abstract The trifluoroethanol-dependent induction of the nonnative α -helical form of β -lactoglubulin has been studied by circular dichroism spectroscopy. Data analysis is performed by factor analysis and multivariate curve resolution. An intermediate form in the induction of the α -helical form of the β -lactoglobulin has been identified at low TFE concentration. By application of an alternating least-squares algorithm, the CD spectrum corresponding to the intermediate form has been resolved. The deconvolution of this CD spectrum shows a secondary structure content more in agreement with the one predicted from the amino acid sequence than the secondary structure of the helical form obtained at higher TFE concentrations. The additional α -helical content of the form present at higher TFE concentrations could be due to nonspecific interaction of TFE with the polypeptide chain.

Complexation of cadmium by the C-terminal hexapeptide Lys-Cys-Thr-Cys-Cys-Ala from mouse metallothionein: study by differential pulse polarography and circular dichroism spectroscopy with multivariate curve resolution analysis

Abstract The cadmium-binding properties of the C-terminal hexapeptide of mouse metallothionein I, Lys-Cys-Thr-Cys-Cys-Ala, were studied by circular dichroism spectroscopy (CD), differential pulse polarography (DPP) and 113 Cd-nuclear magnetic resonance (NMR). The structure of the multiple cadmium binding sites could not be determined by 113 Cd-NMR because of the insolubility of the Cd–peptide samples at the high concentrations required for NMR. Therefore, alternative approaches were used: CD and DPP. The data were analyzed using a multivariate curve resolution (MCR) approach, based on factor analysis techniques, which allows the identification of the signal corresponding to different metal ions bound in different chemical environments. The CD study confirmed that the binding of Cd 2+ induces important conformational changes in the structure of the peptidic complex, including the formation of a binuclear cluster. The DPP results obtained at various Cd 2+ -to-peptide concentration ratios and pH values, under conditions where electrode adsorption is low, if not negligible, indicated the formation of different Cd 2+ –peptide complexes, and a scheme for the electrochemical reduction of the complexed Cd 2+ ions is proposed. These results show that the application of MCR to complex data, such as those from DPP, allows to reach valuable information which is not possible to be obtained by univariate approaches.

Zinc-binding properties of the C-terminal hexapeptide Lys–Cys–Thr–Cys–Cys–Ala from mouse metallothionein: analysis by differential pulse polarography and multivariate curve resolution

Abstract The zinc-binding properties of the C-terminal hexapeptide of mouse metallothionein I, Lys–Cys–Thr–Cys–Cys–Ala, were studied by differential pulse polarography (DPP) with a recently proposed multivariate curve resolution (MCR) method, based on factor analysis techniques, which allows the identification of the signal corresponding to different metal ions bound in different chemical environments. From DPP results obtained at different Zn 2+ -to-peptide concentration ratios and at different pH values, the formation of different Zn 2+ –peptide complexes is deduced, and a scheme for the electrochemical reduction of the complexed Zn 2+ ions is proposed. Two complexes have been detected: the first one is formed at low Zn-to-FT concentration ratios and shows a 1:2 Zn:FT stoichiometry; at higher ratios a second Zn 2+ is incorporated yielding a Zn 2 FT 2 complex which probably contains a binuclear cluster.

Combined Use of Differential Pulse Polarography and Multivariate Curve Resolution: As Applied to the Study of Metal Mixed Complexes of the Metallothionein Related Hexapeptide

The complexation of the hexapeptide Lys-Cys-Thr-Cys-Cys-Ala (FT), peptidic fragment {56–61} of mouse liver metallothionein MT I, in the presence of both Cd(II) and Zn(II) is investigated as a model to understand the metal-binding properties of natural MTs, in which Cd and Zn are simultaneously present. The influence of Cd(II) or Zn(II) on the complexing behavior of FT with Zn(II) or Cd(II), respectively, is studied using the combined techniques of differential pulse polarography and multivariate curve resolution with alternating least square optimization (MCR-ALS). MCR-ALS allows both the unitary voltammograms for each electrochemical process and the concentration profiles of the different species present in the system to be obtained during the experiment, thereby providing a rough picture of the processes involved. Our results allow us to propose the formation of Cd2ZnFT2 and CdZn2FT2 complexes.

A Chiral Co(II)-Thiolate Clusters Determination in Peptides by Circular Dichroism-Multicomponent Analysis

Metallothioneins (MT) are a class of proteins with a high content in cysteine, which provides them with an especial ability to bind metal ions which is the responsible of its physiological roles like the sequestering of toxic metals1The metal-thiolate stoichiometry and the structure of the protein are dependent on the size, charge and coordination geometry of the bound metal. The magnitude of the sulphur-to-metal charge transfer transitions has been known by analyzing the structures of the metalthiolate cores by spectroscopic techniques. Circular dichroism (CD) has provided great information on stoichiometries and metal coordination geometries, probing the chirality of the metal thiolate clusters in the protein2–3. The structure is altered when the metal coordination geometry changes due to changes in the metal-to-thiolate ratio. Therefore, it is possible to consider several structures depending on the metal concentration. This fact hinders the analysis of the CD spectra due to the complexity of the response. In this context the application of factor analysis multivariate techniques is very useful. Glutathione (GSH) and the C-terminal fragment Lys-Cys-Thr-Cys-Cys-Ala of mouse liver MT I (FT) are choosen as models for thiolate ligands to check the possibilities a multicomponent resolution method4 (MCR) to detect the number of different structures for the Co(II)-GSH and Co(II)-FT complexes.

Application of the Factor Analysis to the Monitoring of the Changes in Protein Structure by Circular Dichroism

Circular dichroism (CD) spectroscopy is a useful tool for the detection of changes in both the secondary and tertiary structure of proteins induced by processes such as, protein folding. Despite the big amount of information that the CD spectra contain, these changes are monitored using a selected and limited number of wavelengths. The choosing of these wavelengths is made as a function of an a priori hypothesis about the nature of the change (i.e the loss of a-helix secondary structure is monitored by the intensity of the band at 222 nm). In this work, we propose the use of Factor Analysis techniques for the study of the structural changes in proteins. This methods is based on the use of a family of computational and statistical techniques concerned to the isolation of sources of variation in an experimental data set, without any a priori assumption about the contribution of the different actors [1]. Among these techniques, a new multivariate curve resolution method has been shown to be a powerful method for the study of the conformational changes using spectrophotometric techniques [2]. Here we study the possibilities of the multivariate curve resolution method for the CD monitoring of the structural changes in the formation of the pH-induced molten globule state of α-lactalbumin.