M.M. Correia dos Santos

Electrochemical studies on small electron transfer proteins using membrane electrodes

Membrane electrodes (ME) were constructed using gold, glassy carbon and pyrolytic graphite supports and a dialysis membrane, and used to study the electrochemical behavior of small size electron transfer proteins: monohemic cytochrome c522 from Pseudomonas nautica and cytochrome c533 as well as rubredoxin from Desulfovibrio vulgaris . Different electrochemical techniques were used including cyclic voltammetry (CV), square wave voltammetry (SW) and differential pulse voltammetry (DP). A direct electrochemical response was obtained in all cases except with rubredoxin where a facilitator was added to the protein solution entrapped between the membrane and the electrode surface. Formal potentials and heterogeneous charge transfer rate constants were determined from the voltammetric data. The influence of the ionic strength and the pH of the medium on the electrochemical response at the ME were analyzed. The benefits from the use of the ME in protein electrochemistry and its role in modulating the redox behavior are analyzed. A critical comparison is presented with data obtained at non-MEs. Finally, the interactions that must be established between the proteins and the electrode surfaces are discussed, thereby modeling molecular interactions that occur in biological systems. # 2002 Elsevier Science B.V. All rights reserved.

Flow amperometric determination of pharmaceuticals with on-line electrode surface renewal

In this article, a flow system developed for the amperometric determination of a great variety of pharmaceuticals that are known to lead the rapid poisoning of the working electrode surface is described. The referred system was made up of two parallel flow channels that shared the voltammetric detector of tubular configuration, whose movement in the manifold followed the concept of multi-site location of detector. In this way, after each measurement, the conditioning of the working electrode was possible through the passage by its surface of a regeneration solution without implying the alteration of the carrier that flowed in the analytical channel of the manifold. The methodology proposed was evaluated through the determination of two drugs belonging to two distinct therapeutic groups: an antihypertensive (diltiazem) and a non-steroid anti-inflammatory (nimesulide). The results obtained after evaluation of various pharmaceutical formulations on the Portuguese market were in the case of diltiazem compared with those supplied by the reference US Pharmacopoeia XXIV method, with no statistically significant differences having been observed for a confidence interval of 95%. In the case of nimesulide, since no official reference method exists, a series of recovery experiments were proceeded with and a mean value of 101.1% with a R.S.D. of 0.7% was obtained.

Voltammetric studies of purine bases and purine nucleosides with copper

Anodic stripping voltammetry with a hanging mercury drop electrode has been used to investigate the interaction of cooper with the purines adenine, hypoxanthine, xanthine and purine nucleosides adenosine, guanosine and inosine at an ionic strength of 0.1 M in KNO3 and in the pH range 3.5–5.5. In all cases stabilisation of copper(I) occurs suggesting that the oxidation of copper(0) in the presence of excess ligand proceeds in two one-electron steps. Adsorption onto the electrode has been analysed and conditions where this is negligible were chosen for complexation studies. From the shift of the peak potential corresponding to Cu(0)/Cu(I) oxidation with increasing ligand concentration the stoichiometry of the complexes and their formation constants have been determined. The values obtained are discussed in terms of the ligand structure.

Flow injection-assisted optical sensor for determination of iron(II) and iron(III) in natural water

Abstract An approach for speciation of iron (as Fe(II) and Fe(III)) based on integration of retention of the Fe(III)-SCN complex with detection using a conventional spectrophotometer is proposed here. The device (namely, a flow-cell packed with an exchange resin) has been coupled to a flow-injection manifold with inner-coupled injection valves which enables discrimination between Fe(III) and Fe(II) taking advantage of a redox minicolumn housed in the loop of one of the valves. The method thus proposed has good selectivity with a determination limit of 80 μgl −1 and affords a determination range of 80–500 μgl −1 for Fe(II) plus Fe(III). Application to synthetic and real samples containing both the oxidation states of iron lead to average recoveries of 103% and 101% for Fe(II) and total iron ( Fe ( II )+ Fe ( III )), respectively, thus showing the usefulness of the overall approach.

Electroanalytical chemistry of cadmium complexes of amino acids at the ionic strength of seawater (0.70 M NaCLO4): Part II

Abstract The complexation of cadmium with naturally occurring amino acids was studied at the ionic strength of seawater (0.70 M in sodium perchlorate medium) and at 25±0.1°C by differential pulse polarography and potentiometry with a glass electrode. The Miniquad program was used to analyse and refine the results of the potentiometry. Although the stability of the complexes is very similar since the chelate groups of the ligands are the same, the rate of dissociation of the complexes can be different. The kinetic aspect has been studied by cyclic voltammetry which is under the influence of the type of adsorption. When this is strong the diffusion and adsorption peaks are well separated and, in principle the rate constant of dissociation of the complex can be determined. If there is weak adsorption the diffusion peak is distorted by adsorption and kinetic studies cannot be done. the adsorption of the ligands themselves has been studied by ac polarography at a 90° angle phase and it has been noticed that ligands with π bonds, sulphur groups or with long hydrocarbon chains are more adsorbed than those without.

VOLTAMMETRIC BEHAVIOUR OF COPPER COMPLEXES WITH CYTOSINE AND ITS NUCLEOSIDE

Abstract The interaction of divalent metal ions such as copper ions with basic constituents of nucleic acids may help to gain an insight into the mechanisms with DNA itself. In this work the complexation of copper by pyrimidine bases and their nucleosides, namely cytosine and cytidine, is analysed at the ionic strength of 0.10 M and in the pH range 3.5–6.5, using anodic stripping voltammetry with the hanging mercury drop electrode. In the presence of cytosine and cytidine, stabilisation of copper(I) oxidation state occurs due to complex formation, and so copper(II) redox reaction proceeds in two one-electron steps. From the peak potential shifts with increasing ligand concentration, the stoichiometry and formation constants of the complexes with copper(II) and copper(I) have been determined. The values obtained were analysed in terms of ligand structure.

Cadmium complexes of aminoacids in seawater conditions

Abstract Potentiometry and differential pulse polarography were used to determine stability constants of cadmium complexes with alanine, serine, valine and glutamic acid at 20 and 25±0.1°C in 0.70 M sodium perchlorate to simulate the ionic strength of seawater. The stability constants are of the same order of magnitude because the chelate groups of the ligands are the same. However, the complexes with alanine, serine and valine are completely labile in polarographic terms but the dissociation of the complex ML with glutamic acid is slower. The rate constant of this reaction has been determined by cyclic voltammetry. The importance of the kinetic point of view in speciation studies is emphasized.

Study of CE mechanisms by square wave voltammetry : Cd(II) + nitrilotriacetic acid and Cd(II) + aspartic acid systems

Abstract The use of square wave voltammetry (SWV) at a hanging mercury drop electrode to study a chemical reaction preceding a reversible electron transfer (CE mechanism) is reported. Two cases were analysed: cadmium(II) + nitrilotriacetic acid and the cadmium(II) + aspartic acid systems. In both situations the homogeneous reaction in buffered solutions and in the presence of an excess of ligand was considered to be first order. Experimental data were analysed in terms of theory of SWV for kinetic systems. Rate constants of dissociation of the complexes of cadmium with nitrilotriacetic acid and aspartic acid were determined using a numerical procedure based on a one variable fit. The results obtained are in good agreement with published values where other techniques have been used, in particular cyclic voltammetry.

Determination of stability constants of quasi-reversible systems: Copper chlorocomplexes in seawater conditions

Abstract The dc polarography with dropping mercury electrode and dc voltammetry with a rotating wire platinum electrode were used to obtain stability constants of Cu(I) and Cu(II) chlorocomplexes at 20±0.1°C and at the ionic strength 0.70 M (NaClO4+NaCl). The systems are quasi-reversible and two calculation methods have been used to determine the formation constant of the Cu(I) chlorocomplex. The transfer coefficient and the rate constant have also been determined. The use of the rotating wire platinum electrode has been discussed.

Electrochemical studies on c-type cytochromes at microelectrodes

The aim of this work is to use microelectrodes as a current approach for the study of unmediated electrochemistry of redox proteins. An electrochemical study of monohemic cytochromes c552 from Pseudomonas nautica 617, cytochrome c553 from Desulfovibrio vulgaris and horse heart cytochrome c is presented at inlaid disk microelectrodes of platinum, gold and carbon. Different electrochemical techniques were used such as linear scan, differential pulse and square wave voltammetry. The electrochemical response was also analysed at conventional size (macro) electrodes for comparison. In all situations a promoter was used. The electrochemical behaviour was evaluated in terms of kinetics of the electrode processes and the formal potentials determined. Diffusion coefficients were also calculated from the voltammetric data. A critical comparison of the results obtained is carried out and the advantages of microelectrodes for electrochemical studies of metalloproteins are pointed out.