RESUMEN
X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.
RESUMEN
The protective effect of the bisphosphonate drug xydiphone (K,Na-ethidronate) on membrane-bound enzyme damaged by lead ions has been studied. A protein/lipid film of Ca-ATPase/phosphatedylethanolamine deposited on a silicon substrate was used as a model system. The position of lead ions within the molecular film before and after the xydiphone treatment was determined using the total-reflection X-ray fluorescence method. This technique is based on the simultaneous measurement of the X-ray reflection and the yield of the fluorescence radiation excited by X-ray inelastic scattering. The possibility of directly locating lead ions is the main advantage of this approach. Xydiphone has been found to effectively eliminate lead ions that have been incorporated into Ca-ATPase molecules during a preliminary incubation in lead acetate solution. The lead ions that were bound at the sites of the Ca-ATPase attachment to the phospholipid monolayer have proved to be inaccessible for xydiphone. A preliminary incubation of Ca-ATPase in the xydiphone solution precluded the incorporation of lead ions into the protein.
