Evaluation of the protein concentration in enzymes via the determination of sulphur by TXRF

Total reflection x-ray fluorescence spectrometry (TXRF) offers many advantages for the identification of trace elements in biological samples like enzymes, tissues or plants. Without any preliminary treatment elements may be determined with high accuracy especially transition metals like Fe, Ni, Cu, Mo and the alkaline earth metal Ca. A further aspect of the investigation of enzymes is the simple and simultaneous determination of light elements. Especially sulphur is of interest. The element sulphur exists mainly in the two amino, acids methionine and cysteine as well as in iron-sulphur clusters and may be used for an easy and simultaneous calculation of the protein concentration. Hence the quantitative determination of sulphur by TXRF allows a cross-check regarding of the conventional quantitative determination of protein concentration by, for example, the Lowry method. On the basis of three enzymes of different origins and molecular weights the presentation will show the influence of the bio-organic matrix and different buffer media on element determination by TXRF. As is already known the influence of the matrix on the detection of light elements is stronger than on transition metals. It can be discussed whether layer thickness and layer effects of the drying residues (characterization by SEM and thickness profilometer (ALPHA-step)) and / or self absorption effects as well as the excitation are of significance. The results indicate that with enzymes of low molecular weight a reliable determination of sulphur is possible whereas those with higher molecular weights gave poorer results on account of the matrix effects described. (author)