| It is estimated that 1/4 to 1/2 of all proteins contain metals, which may be used to carry out enzymatic catalysis or serve structural roles. The determination of the proteins which bind metals, and which metals are bound (the metallome) is important in the determination of the roles metals play in biology, and how the metals are regulated. Due to the large number of proteins expressed in a proteome, a high-throughput method for metallome determination is desirable. X-ray fluorescence (XRF) is a sensitive method for determining the presence of metals, and is well-suited to studies of the metallome. Two XRF schemes were tested for metallome investigation. In one, the excitation energy was scanned across the absorption edge, while in the other, the excitation energy was fixed and the fluorescence energy was scanned. Both detection schemes were able to give detection limits in the micromolar range, using automated data analysis programs developed for high-throughput studies. The excitation scans were analyzed using both edge jump values and the peak areas of the first derivative to determine the metal concentrations. The fluorescence scans gave low detection limits (6--15 muM) for metals when the excitation energy was within approximately 1700 eV of the metal's absorption edge, with the sensitivity falling off rapidly beyond that point. Detection limits with excitation energies in the range of 6 to 11 keV were simulated for the transition metals chromium through zinc, based upon results from the fluorescence scans. These simulations showed the best response for most metals to be just above the absorption edge, as would be expected. However, each element had a different decrease in detection limits as the excitation energy was increased beyond the absorption edge, due to the different characteristics of each metal's fluorescence energy. Further information was obtained on metallopeptides with extended x-ray absorption fine structure (EXAFS), which was able to give more detailed ligand information on biological metal sites for zinc and de novo designed peptides which bound arsenic. |
