DETERMINATION OF METAL-COFACTORS IN RESPIRATORY-CHAIN COMPLEXES BY TOTAL-REFLECTION X-RAY-FLUORESCENCE SPECTROMETRY (TXRF)

Total Reflection X-Ray FIuorescence Spectrometry (TXRF) offers many ad vantages for the detection of trace elements in enzymes as compared to other well known analytical techniques like flame-AAS or TCP-AES beca use of the significantly smaller amounts of sample required. Without a ny decomposition, elements like Fe, hii. Cu, Zn, Mn and Mo could be de termined with high accuracy, in spite of the large bio-organic matrix. Besides the metals also sulfur can be determined in protein samples. The two terminal oxidases, cytochrome c oxidase and quinol oxidase, is olated from the soil bacterium Paracoccus denitrificans, were transfer red from their usual salt buffer into a solution of 100 mmol/L tris(hy droxymethyl)aminomethane (TRIS) acetate containing an appropriate dete rgent. By this procedure an improved signal/noise ratio is attained. T he data for cytochrome c oxidase are in good agreement with values obt ained by ICP-AES. Further results of quinol oxidase, which has differe nt element ratios, also fit the expected values. The investigations le ad to the conclusion that the method is well suited for the quantitati ve determination of metals in enzymes. and in particular their molar r atios, and requires only small amounts of the biological sample withou t any extensive pretreatment.