Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC‐PAGE): An Efficient Method for Isolating Cadmium Cofactors in Biological Systems

Abstract

A new electrophoretic method [preparative native continuous polyacrylamide gel electrophoresis (PNC‐PAGE)] was developed to isolate cadmium cofactor‐containing proteins in biological systems. For this purpose Arabidopsis cytosol was subjected to gel permeation chromatography (GPC) and high molecular mass cadmium proteins (MW ≈ 200 kDa) in a GPC fraction of this plant were isolated by PNC‐PAGE. Furthermore plant cytosol was directly separated by this method. The cadmium concentrations in all GPC and PAGE fractions were determined by GF‐AAS using matrix modifier. As electrophoresis buffer of the PAGE method a Tris–HCl buffer (20 mM Tris, 1 mM NaN₃, pH 10.00) was used. The gel (degree of polymerization of the polyacrylamide: 4%; gel length: 4 cm) was polymerized for 69 hr. By these procedures it was revealed that the high molecular mass cadmium proteins of Arabidopsis can be detected in quantitative amounts by using PNC‐PAGE. It could be shown that the chemical structure of the cadmium proteins did not change under these PAGE conditions. PNC‐PAGE is supposed to be an efficient method for isolating other metal cofactors such as Zn, Cu, Ni, Pd, Co, Fe, Mn, Pt, Cr, and Mo and might be suitable for subsequent structural determinations of metalloproteins present in PAGE fractions.

Keywords:

• Cadmium cofactors
• Arabidopsis
• PNC‐PAGE

Acknowledgments

I am grateful to Prof. Dr. U. Schurr, Prof. Dr. K. Günther, B. Muktiono, Dr. U. Katscher, Dr. M. von Sparr, Research Centre Jülich, my wife Claudia and our little son Noah.