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 of fers many advantages for the determination of trace elements in enzyme s, as compared to other well known analytical techniques such as flame atomic absorption spectrometry or inductively coupled plasma atomic e mission spectrometry (ICP-AES), because of the significantly smaller a mounts 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 re ductase 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-buff er 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 reduct ase; was found to contain copper as a hitherto existing unknown cofact or. The enzyme contains a stretch of amino acids that are typical of c opper proteins and thus confirm the presence of this element. Furtherm ore, the data concerning cytochrome c oxidase from Paracoccus denitrif icans 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 dete rmination of metals in enzymes, in particular their molar fractions, a nd requires only small amounts of the biological sample without any ex tensive pretreatment. (C) 1997 Elsevier Science B.V.