Axel Wittershagen

Determination of metal-cofactors in enzyme complexes by total-reflection X-ray fluorescence spectrometry

The determination of metal-cofactors and their molar concentrations is an important requirement for the characterisation of metalloproteins and a challenge regarding the capabilities of trace analytical methods. In this respect, total-reflection X-ray fluorescence spectrometry offers many advantages for the determination of trace elements in enzymes, as compared to other well known analytical techniques such as flame atomic absorption spectrometry or inductively coupled plasma atomic emission spectrometry (ICP-AES), because of the significantly smaller amounts of sample required. Without any decomposition, elements like P, S, Fe, Ni, Cu, Zn, Mn and Mo could be determined with high accuracy, in spite of the large bio-organic matrix. The enzymes (polysulphide reductase and hydrogenase of the rumen bacterium Wolinella succinogenes, and the cytochrome c oxidase and quinol oxidase of the soil bacterium Paracoccus denitrificans) were transferred from their usual salt-buffer into a solution of 100 mmol l−1 tris(hydroxymethyl)aminomethane (tris)-acetate containing an appropriate detergent. By this procedure, an improved signal-to-noise ratio is obtained. The polysulphide reductase was found to contain copper as a hitherto existing unknown cofactor. The enzyme contains a stretch of amino acids that are typical of copper proteins and thus confirm the presence of this element. Furthermore, the data concerning cytochrome c oxidase from Paracoccus denitrificans are in good agreement with published values obtained by ICP-AES. Also, results from measurements with the quinol oxidase from the same bacterium agree with the expected values. The investigations lead to the conclusion that the method is well suited to the quantitative determination of metals in enzymes, in particular their molar fractions, and requires only small amounts of the biological sample without any extensive pretreatment.

Trace element determination in drugs by total-reflection X-ray fluorescence spectrometry

Abstract The capability of total-reflection X-ray fluorescence spectrometry (TXRF) for the determination of trace elements in drugs is described. Various samples of lecithin, insulin, procaine and tryptophan of different origin were investigated. The element concentrations provide element fingerprints which offer the possibility to discriminate between different batches of the analysed substances originating from different production or purification processes. TXRF facilitates the characterization of such samples without extensive pre-treatment, and provides fast multi-element determination of elements with atomic numbers 14

MUTATIONS IN THE CA2+ BINDING SITE OF THE PARACOCCUS DENITRIFICANS CYTOCHROME C OXIDASE

Recent structure determinations suggested a new binding site for a non‐redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site‐directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild‐type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp‐477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Asp‐193 and Glu‐218 of subunit II are involved in the Mn2+‐binding of Paracoccus denitrificans cytochrome c oxidase

Cytochrome c oxidase contains a binding site for a non‐redox‐active metal at the interface of subunits I and II, usually a magnesium ion. In Paracoccus denitrificans oxidase, typically 20% may be replaced by manganese, using standard growth media. Site‐directed mutants were constructed in subunit II (D193N and E218Q), and the isolated enzymes analyzed by total‐reflection X‐ray fluorescence spectrometry and EPR. Both mutants show a strong reduction of the manganese stoichiometry and a diminished electron transfer activity, demonstrating that D193 and E218 are involved in the binding of a manganese/magnesium ion in this site.

Total-reflection X-ray fluorescence study of electrochemical deposition of metals on a glass-ceramic carbon electrode surface☆

Abstract The features of electrochemical deposition and co-deposition of copper, cadmium and lead from aqueous solutions on disc glass-ceramic carbon (GCC) electrode surfaces were studied by total-reflection X-ray fluorescence analysis (TXRF). This method was found to be highly sensitive to the varieties of electrodeposit morphology and depth distribution of elements on the electrode surface. It allows identification of the mechanisms of metal nucleation and growth of thin film electrodeposits. The results of the TXRF study are in good agreement with the recent data of a number of spectroscopic and microscopic methods of solid surface analysis. The polished GCC was shown to be an excellent material for preparation of the sample carriers for TXRF analysis.

Total-reflection X-ray fluorescence spectrometry, a powerful tool for semiquantitative analysis of archaeological glass samples

Abstract The chemical composition of old glasses provides considerable information about the knowledge and technology of our ancestors and about their ability and developments in the art of glass manufacturing. Two valuable exhibit items from the Hessisches Landesmuseum Kassel, the “Reichsadlerhumpen” and the “Flacon”, have been analysed for the first time. The analysis was performed semiquantitatively by total-reflection X-ray fluorescence spectroscopy (TXRF). The micro samples were taken carefully from the bottom to minimize any damage. In this respect, it is very useful that TXRF requires only small amounts of sample. The semiquantitative analysis of the investigated “Reichsadlerhumpen” compared with other results supports the hypothesis of Bohemian origin. The TXRF analysis of the “Flacon” reveals a very low content of Ca, and therefore the hypothesis can be confirmed that glass containing such a low Ca concentration has the tendency to deteriorate (glass corrosion). The analysis demonstrates that TXRF is a powerful tool for a nearly non-destructive semiquantitative analysis of archaeological glass finds. TXRF is suitable for achieving a rapid analysis and an overview of elements with Z >14 and provides information on element ratios.