QUANTIFICATION BY PIXE OF METALLIC SITES IN PROTEINS SEPARATED BY ELECTROPHORESIS

Electrophoresis on polyacrylamide gel (PAGE) is widely used in life sc iences to determine the molecular weight of proteins in solution by se parating them into different bands. By coupling electrophoresis and Pa rticle Induced W-ray Emission (PIXE), the nature and the quantity of m etals contained in proteins can be investigated. After the electrophor esis, 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 t he determination of the metals contained in an electrophoretic band. T he metal content in each band is obtained by comparing the characteris tic X-ray peak area with those obtained with polyacrylamide gels doped with the same metal. Finally, the relative concentration of each prot ein is determined by densitometry in order to compute the protein/meta l ratio. An example of metallic site determination is presented. This procedure seems to be a very useful multielementary method for the det ermination of the metal amounts inside proteins after their separation by electrophoresis. Furthermore it allows to check if metals remain b ound to proteins. (C) 1998 Elsevier Science B.V.