Evaluation of the protein concentration in enzymes via determination of sulfur by total reflection X-ray fluorescence spectrometry limitations of the method

Ž . Total reflection X-ray fluorescence spectrometry TXRF offers many advantages for the identification of trace elements in biological samples like proteins, enzymes, tissues or plants. Because of difficult and time consuming isolations and cleaning procedures enzyme samples are often available in small amounts only. Using TXRF without any preliminary treatment, a ‘screening’ of such samples to determine the element composition is of interest and importance. Transition metals like Fe, Ni, Cu, Mo and the alkaline earth metal Ca may be determined with high accuracy. A further aspect of the investigation of enzymes is the simple and simultaneous determination of light elements. Sulfur, especially, is of interest. This element is a component of two amino acids, methionine and cysteine, and of iron sulfur clusters and may be used for easy and simultaneous calculation of the protein concentration. Hence quantitative determination of sulfur by TXRF allows a cross-check regarding of conventional quantitative determination of protein concentration by, e.g. the Lowry method. On the basis of two selected enzymes of different origins and molecular weights this paper will demonstrate the influence of bio-organic matrix and different buffer media on sulfur determination by TXRF. The influence of layer thicknesses of the dry residues and absorption or scattering effects will be discussed. The results indicate that in enzymes with low molecular weights and minor amounts of buffer components a reliable determination of sulfur is possible. By contrast, for enzymes stored in higher buffer concentrations poorer results are given on account of the matrix effects described. 2001 Elsevier Science B.V. All rights reserved.