Quantification by PIXE of metallic sites in proteins separated by electrophoresis

Electrophoresis on polyacrylamide gel (PAGE) is widely used in life sciences to determine the molecular weight of proteins in solution by separating them into different bands. By coupling electrophoresis and Particle Induced W-ray Emission (PIXE), the nature and the quantity of metals contained in proteins can be investigated. After the electrophoresis, the gel is dried and each track is scanned with a 2.5 MeV proton beam which induces X-ray emission. Analysis of these spectra allows the determination of the metals contained in an electrophoretic band. The metal content in each band is obtained by comparing the characteristic X-ray peak area with those obtained with polyacrylamide gels doped with the same metal. Finally, the relative concentration of each protein is determined by densitometry in order to compute the protein/metal ratio. An example of metallic site determination is presented. This procedure seems to be a very useful multielementary method for the determination of the metal amounts inside proteins after their separation by electrophoresis. Furthermore it allows to check if metals remain bound to proteins. (C) 1998 Elsevier Science B.V.