Michael Ottnad

X-ray photoelectron spectroscopic studies of copper- and zinc-amino acid complexes and superoxide dismutase

Abstract X-ray photoelectron spectra were recorded to obtain information regarding the oxidation state of some metallic and non-metallic elements in erythrocuprein. Investigations of various Cu and Zn complexes and salts show marked differences between the binding energies of ionic and complexed bound metals and their ligands. From the intensities of the X-ray photoelectron spectra of the Cu 2p 3 2 and Zn 2p 3 2 levels of erythrocuprein, it can be concluded that Cu is located at the surface of this metalloprotein, whereas Zn must be bound more inside the molecule. X-ray photoelectron spectroscopy promises to be a most valuable new analytical technique for studies on metalloproteins. It is particularly suitable for those metal ions which cannot be detected by electron spin resonance or Mossbauer spectroscopy. In addition to the investigation of metal ions in proteins, other elements, e.g. S present in relatively low concentrations, can be detected.

Quantitative determination of 0-(β-hydroxyethyl)-rutosides in human blood after intravenous and oral administration by circular dichroism

SummaryA new method is described for the quamtitative spectroscopic detection of hydroxyethylated rutosides in body fluids. This sensitive and specific technique differs in many aspects from the common methods used in pharmacokinetics and drug metabolism. No chromatographic procedures or radioactive labelling are required and the concentration of drugs can be determined directly in serum or plasma. Human serum shows a positive Cotton effect in the circular dichroism spectrum at about 345 nm, whereas hydroxyethylrutosides exhibit a strong negative circular dichroic absorption at the same wavelength. Therefore standardized circular dichroic measurements of the serum or plasma at 345 nm allow the determination of concentration levels.The application of this technique for the determination of the kinetics after intravenous and oral administration of the drug Venoruton (HR) is described. Reproducible results are obtained after application of 1500 mg i.v. or 4 g per os to male volunteers kept on a flavonoid-free diet. It can be shown that the drug level in blood decreases within 2–4 hours from 5 mg/100 ml at 15 min after the application of 1500 mg i.v. to less than 0.1 mg/100 ml. At least 50% of the administered drug appear in the urine. After oral administration of 4 g HR the blood concentration of Venoruton has a maximum between 6 to 10 hours and the drug can be detected by circular dichroism up to 25 hours in blood, however, evaluation of the data for pharmacokinetics is not possible.

Circulardichroismus- und13C-NMR-Untersuchungen zur Dissoziation von Aminosäuren

The dissociation of amino acids such, as histidine, methionine and of shorter peptides can be studied by pulse Fourier transform.13C-NMR spectroscopy. The pH-dependencies of the13C-signals show directly the changes of the electronic charge densities of all carbon atoms caused by the dissociation process. Additional comparative studies of the pH and temperature dependencies of the CD spectra and the1H-NMR spectra give now better information on the steric and electronic changes due to the dissociation of polyfunctional amino acids and oligopeptides.ZusammenfassungDie Dissoziation von Aminosäuren wie Histidin, Methionin und kleineren Peptiden kann mit Hilfe der Impuls-Fourier-Transform-13C-NMR-Spektroskopie untersucht werden. Aus der pH-Abhängigkeit der13C-Signale lassen sich die durch die Dissoziation verursachten Änderungen der Ladungsdichteverteilung an allen Kohlenstoffen direkt verfolgen. Durch vergleichende zusätzliche Untersuchungen der pH- und Temperaturabhängigkeit der CD-Spektren sowie der1H-NMR-Spektren lassen sich sterische und elektronische Änderungen bei Dissoziationsvorgängen mehrfunktioneller Aminosäuren und Oligopeptide eingehender als bisher untersuchen.

X-Ray photoelectron spectroscopy (XPS) of bovine erythrocuprein☆

Structural aspects and biochemical functions of erythrocuprein have been intensively studied’in the last decades [l] . The metalloprotein consists of two 16 300 molecular weight subunits [2,3] and contains two g-atoms of copper and zinc per 32 600 g of protein. McCord and Fridovich [4] successfully proposed an enzymic function, namely the enzymic catalysed disproportionation of 0,. into H,O, and 0,. This biochemical function was critically examined and another reactivity, the quenching of singlet oxygen was presumed the main role of this protein [S] . Nevertheless, the ubiquity of erythrocuprein in many aerobic cells and its reactivity in the biochemistry of oxygen suggests a most important and possible essential function. In this context we were especially interested in the two metals copper and zinc. At the moment our knowledge of the binding situation of these metallic components is rather poor. Thus we thought it valu. able to receive some information regarding their location in the protein portion. Due to the biochemical reactivity it was expected that copper, for the least, should be readily accessible to the substrate. X-Ray photoelectron spectroscopic measurements gave rise to the conclusion that indeed the cupric ion must probably be located on the surface of the protein portion while Zn*+ appears to be bound in the inner

Circulardichroismus des disulfidchromophors in S-alkylthio-L-cysteinen und L-glutathion

Zusammenfassung Der inharent dissymmetrische Disulfid-Chromophor ruft in Molekulen, die dem Cystin verwandt sind und eine Disulfidgruppierung C-S-S-C mit P- oder M-helikaler Anordnung besitzen, zwei optisch aktiveUbergange mit entgegengesetztem Vorzeichen bei 250 nm hervor. DerUbergang dieses intrinsisch optisch aktiven Chromophors zeigt eine starke Abhangigkeit der Rotationsstarke vom Torsionswinkel ϕ. Die offenkettigen S- Alkylthio-L-cysteine zeigen grosse Temperaturkoeffizienten ihrer beiden negativen Cotton-Effekte bei 250 nm und 200 nm im Bereich von 0°C bis 100°C in saurer wassriger Losung. Fur den S-S-Chromophor von oxidiertem L-Glutathion wird eineahnlich ausgepragte Temperaturabhangigkeit gefunden. Demgegenuber zeigen die Carbonylubergange dieser Molekule lediglich den normalen Kauzmann-Eyring-Effekt. Die Grosse und Lage der Disulfid-Cotton-Effekte wird stark von den Dissoziationsgleichgewichten beiinflusst, wobei mit zunehmendem pH bathochrome Verschiebungen der 250 nm-Disulfidbande gefunden werden.

Röntgen-Photoelektronen-Spektroskopie von Metallen in Aminosäurekomplexen und Proteinen

X-ray photoelectron spectroscopy gives information about the oxidation states and the ligands of metal ions in metalloproteins. To interpret the spectra of the metalloenzymes the photoelectron spectra of amino acid complexes were recorded. Drastic differences were noted for the binding energies of Cu and Co in the complexes and in the proteins. This is probably due to metal-metal interactions in the investigated erythrocupreins. Sulphur-containing compounds are very suitable for X-ray photoelectron spectroscopic measurements. It could be shown that sulphur is not involved in the binding of the metal ions.ZusammenfassungDie Röntgen-Photoelektronen-Spektroskopie liefert Informationen über die Oxidationsstufen und Liganden von Metallionen in Metalloproteinen. Zur Interpretation der Spektren der Metalloenzyme wurden die Photoelektronenspektren von Aminosäurekomplexen aufgenommen. Für die Cu- und Co-Verbindungen ergaben sich drastische Unterschiede der Bindungsenergien der Komplexe verglichen mit den Werten in den Proteinen. Dies ist wahrscheinlich auf Metall-Metall-Wechselwirkungen in den untersuchten Erythrocupreinen zurückzuführen. Schwefelhaltige Verbindungen sind zur Untersuchung mit der Röntgen-Photoelektronen-Spektroskopie besonders geeignet. Es konnte gezeigt werden, daß Schwefel nicht an der Bindung zum Metall beteiligt ist.