Ole Farver

Interfacing capillary electrophoresis with inductively coupled plasma mass spectrometry by direct injection nebulization for selenium speciation

A demountable direct injection high efficiency nebulizer operating at low sample uptake rates was developed and used for coupling of capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS). When the nebulizer was used for continuous sample introduction, detection limits of 20 and 1xa0ngxa0L−1 were obtained for 82Se and 103Rh, respectively, at sample uptake rates of 10–30xa0μLxa0min−1, based on three times the standard deviation of blank solution (3σb, n = 10). The nebulizer was used as part of the interface for coupling of CE with ICP-MS and applied for speciation of aqueous selenium standards. The interface was operated in the self-aspirating mode with a sheath liquid uptake of 10xa0μLxa0min−1. The CE-ICP-MS system resulted in baseline separation of selenate, selenite, selenocystine and selenomethionine within a total analysis time of 5.4xa0min. Detection limits were in the sub µgxa0Sexa0L−1 range, corresponding to absolute detection limits in the range 25–125xa0fg selenium. Repeatability (n = 6) expressed as relative standard deviations with respect to migration times, peak heights and peak areas were better than 1.6, 6.7 and 6.0%, respectively.

Human Ceruloplasmin INTRAMOLECULAR ELECTRON TRANSFER KINETICS AND EQUILIBRATION

Pulse radiolytic reduction of disulfide bridges in ceruloplasmin yielding RSSR− radicals induces a cascade of intramolecular electron transfer (ET) processes. Based on the three-dimensional structure of ceruloplasmin identification of individual kinetically active disulfide groups and type 1 (T1) copper centers, the following is proposed. The first T1 copper(II) ion to be reduced in ceruloplasmin is the blue copper center of domain 6 (T1A) by ET from RSSR− of domain 5. The rate constant is 28 ± 2 s−1 at 279 K and pH 7.0. T1A is in close covalent contact with the type 3 copper pair and indeed electron equilibration between T1A and the trinuclear copper center in the domain 1–6 interface takes place with a rate constant of 2.9 ± 0.6 s−1. The equilibrium constant is 0.17. Following reduction of T1A Cu(II), another ET process takes place between RSSR− and T1B copper(II) of domain 4 with a rate constant of 3.9 ± 0.8. No reoxidation of T1B Cu(I) could be resolved. It appears that the third T1 center (T1C of domain 2) is not participating in intramolecular ET, as it seems to be in a reduced state in the resting enzyme.

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1‐methyl‐nicotinamide radical (MNA∗), produced by pulse radiolysis has previously been shown to reduce the CuA‐site of cytochromes aa 3, a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a 3 [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131–2137]. Investigating this process in the cytochrome ba 3 of Thermus thermophilus (Tt), we now show that MNA∗ also reduces CuA with a subsequent ET to the heme b and then to heme a 3, with first‐order rate constants 11 200 s−1, and 770 s−1, respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a 3‐CuB center can be reduced in molecules containing a single reduction equivalent.

The role of the medium in long-range electron transfer

Abstractu2002The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism.

Three-dimensional model of stellacyanin and its implications for electron transfer reactivity

Experimental data were combined with computational methods in constructing a hypothetical three-dimensional model for the blue single copper protein Rhus stellacyanin (St). The known sequence of stellacyanin and its homology with plastocyanin (Pc) were used together with the results of spectroscopic studies of the protein that yielded the current assignment of two histidines, one cysteine and a disulfide sulfur as copper ligands in stellacyanin. By computer graphics and energy minimization the folding of the protein was predicted. The model structure is somewhat less regular than Pc as judged by surface area and energy comparisons, but it is a stable structure. Besides rotation of one imidazole ring the copper site undergoes no change even in the absence of the copper ion and the model shows that the site can be constructed with the four assumed copper ligands without forming a strained system. The structure also indicates that a carbonyl oxygen atom is near the copper, thus the site may have analogy to the Alcaligenes denitrificans azurin (Az) site, although the amino acid sequence is more homologous to that of Pc. The model indicates that aspartate 49, reductively labeled by Cr(III), is near the copper center and homologous to the site labeled by Cr(III) on Pc. Also homologous to Pc is a tyrosine residue adjacent to the aspartate. This tyrosine has been implicated in Pc electron transfer and thus is probably involved in electron transfer reactivity of St as well. The higher reactivity of St with small-molecule redox reagents compared to Az and Pc, may be due to the proximity of the above-mentioned aspartate 49 to the Cu, or the greater exposure of one of the Cu cysteine ligands, in the predicted structure as compared to that in the known Pc and Az structures.

Peroxide binding to the type 3 site in Rhus vernicifera laccase depleted of type 2 copper

Abstract The interaction between hydrogen peroxide and oxidized Rhus vernicifera laccase from which the type 2 copper has been removed, was investigated. For that end, the circular dichroic spectrum of the modified enzyme has been measured in the presence of increasing concentrations of hydrogen peroxide. The characteristic band observed upon binding peroxide to native laccase is also observed for the type 2 copper depleted enzyme. However, there are several quantitative differences in the latter one. First, the intensity is lower and band width is larger. Secondly, from the titrations, it becomes apparent that the affinity for H2O2 is markedly lower than that of the native enzyme. While the affinity for the native enzyme is higher than 108 M−1, it decreases to 1·104 M−1 for the type 2 depleted enzyme.

Magnetic susceptibility study of the laccase-peroxide derivative

Laccases are copper containing oxidases in which the 4 copper ions are bound in 3 distinct sites. Two of these are EPR detectable (type 1 and type 2) while the third site (type 3) most probably consists of an antiferromagnetically coupled Cu(II)-Cu(lI) pair [ 11. The magnetic susceptibility of native oxidized Rhus lactase can be quantitatively accounted for by the EPR detectable copper [2-41. Thus, formulation of the type 3 site as a copper pair requires a lower limit for the coupling, expressed by the exchange integral, J of 250-300 cm-’ [3,4]. We have demonstrated the formation of a high affinity peroxy-lactase complex upon reacting native oxidized enzyme with H202 [5]. Primarily on the basis of the absorption and CD spectra it was proposed that the peroxide binds to the copper pair of the type 3 site [6]. We have shown spectral similarities between the peroxy-lactase complex and intermediates formed during the reoxidation of reduced lactase with O2 [7]. These observations have led us to extend the study of the type 3 site, since the interaction with dioxygen or its derivatives might affect the exchange coupling of this copper pair. Therefore the magnetic properties of peroxy-lactase was measured by means of high resolution NMR technique. Indeed we have observed an increase in the magnetic susceptibility upon peroxide binding which is assigned to a decrease in the antiferromagnetic coupling of the type 3 copper pair.

Peroxide and redox titrations of type 2 copper depleted laccase

Abstract The chemistry of Type 2 copper depleted T2D Rhus laccase has been investigated with regard to the binding of peroxide, and the ability of the enzyme to undergo reduction and reoxidation. Although the peroxide affinity is diminished in the T2D enzyme (10 4 M −1 ) relative to the holo-enzyme ((⩾ 10 8 M −1 ) the actual mode of binding as a Type 3 μ-peroxo complex remains, as indicated by absorption and CD spectral measurements. Anaerobic reductive and reoxidative titrations with hydroquinone and hydrogen peroxide respectively revealed that the Type 3 copper pairwise interaction is disrupted during reduction but can be restored on reoxidation. The concept of separate Type 2 and Type 3 copper redox centers is suggested to be inadequate in view of the loss of functional integrity by the Type 3 site on removal of Type 2 copper.

Hyphenation of CE to ICP-MS and to sheathless electrospray-MS for high sensitivity and selectivity in bioanalysis

SummaryTwo new inferfaces for hyphenation of capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry and sheath less electro spraymass spectrometry, respectively, are described. These hyphenated CE techniques are intended for use in bioanalysis in order to obtain high selectivity and low detection limits combined with the high separation selectivity and efficiency of the CE system.Some test systems as well as two bioanalytical examples are given for illustration of the power of the systems.The absolute limit of detection achieved is in the low femtogram range corresponding to a concentration of about one nanogram per millilitre.

Electron transfer loci on blue copper proteins

Abstract The chemical properties of the Cr(II)/(III) couple has been successfully employed to final affinity label electron transfer loci on redox proteins. Being strong reductants and able to exchange their ligands sphere fast, the Cr(II) ions can coordinate one or more of the surface residues on the protein while reducing it. Since the Cr(III) produced is effectively substitution inert, any protein residues in the coordination sphere of the Cr(II) during the electron transfer will remain bound to the Cr(III) product. Identification of the Cr(III) binding loci has been achieved primarily through proteolytic cleavage of the different labeled proteins. Spectroscopic methods have been useful in corroborating these assignments. Several single blue copper proteins have been examined by the above approach. These include the bacterial electron carriers azurin from Pseudomonas aeruginosa and Alcaligenes faecalis . Plastocyanin (from French bean and poplar tree) which serves as an electron mediator in the photosynthetic apparatus and stellacyanin obtained from the lacquer sap of Rhus vernicifera have all been studied by this method. More recently it has been shown that even the multicentered blue copper oxideue5f8-laccase can be reductively labeled by this procedure. In this latter case, while 3.3 equivalents of Cr(II)aq were required for full reduction of the protein, only 0.7 Cr(III) ions remained bound to laccase after extensive dialysis. This may indicate a single reduction locus for Cr(II) in this protein. The cuprous ions in the Cr(III) labeled plastocyanin, azurin and stellacyanin could be fully reoxidized by inorganic or enzymatic agents. While the original, single Cr(III) ion coordinated to azurin and stellacyanin remains bound through several Cr(II) reduction and reoxidation cycles, one can label plastocyanin with at least two Cr(III) ions in two cycles. In the structure of both plastocyanin and Ps azurin the ‘northern’ end imidazol of His-87 or 117 respectively was considered to be the potential electron transfer site. The analysis of the Cr(III) labeled sites on these two proteins clearly showed that electrons can also be introduced via different loci. These were proposed to proceed via the His 35 region in azurin (Az) and the negative patch on plastocyanin (Pc). To examine whether the latter electron transfer sites are also involved in the biochemical function of these proteins, their reactivities, in the native and Cr(III) labeled forms were compared. For Pc, photoreduction and oxidation by chloroplasts and by photosystem I reaction centers respectively were studied. For azurin the reactions with cytochrome c 551 and Ps . cytochrome oxidase were investigated. It became apparent that the Cr(III) label attenuated the reactivity of both azurin and plastocyanin with only one of their respective partners. This led to the conclusions that on both proteins: (a) There are probably two distinct and physiologically operative electron transfer sites. (b) One of these sites is centered around the respective Cr(III) labeled region. (c) By elimination, the second is at the exposed, homologous imidazol of His-87 or 117 in Pc and Az respectively.