No abstract

This paper describes some recent advances in the methodology of immobilized metal ion affinity gel electrophoresis. Four different ways to incorporate metal chelate ligands in agarose and polyacrylamide-based electrophoresis gels are evaluated, a new polymerizable metal chelating ligand, allyl-2-hydroxy-3-(N,N-dicarboxymethyl)amino-propyl ether, is introduced, and the determination of affinity constants described. The affinities of model proteins (ribonucleases A and B and cytochromes c from different species) for the transition metal chelate iminodiacetic acid-Cu(II) were studied. The results were found to be in agreement with literature data on immobilized metal ion affinity chromatography, and the polymer nature and the different chemistries used influenced the affinity only quantitatively, keeping the basic mechanisms of interaction unchanged.

Oligomerization can endow proteins with novel structural and catalytic properties. The native dimer of bovine seminal ribonucleases (BS-RNase) binds, melts and catalyses the hydrolysis of double-stranded ribonucleic acids 30-fold better than its pancreatic homologue, the monomeric RNase A. Chemically induced oligomers of pancreatic RNase A are also found to show an increase in enzyme activity on double-stranded poly(A).poly(U) (Libonati, M. Bertoldi, M. and Sorrentino, S. (1996) Biochem. J. 318, 287-290) and, therefore, can be considered as potential immunosuppressive and cytotoxic agents. We report here a study on the relationship between surface histidine topography in oligomeric forms of these ribonucleases and their catalytic properties. Subtle changes in structure conformation of both BS-RNase and oligomeric RNase A are shown to result in a modification of the affinity of these proteins toward the immobilized transition-metal chelate, IDA-Cu(II). Because, such conformational change has been shown to correlate with an improvement of the newly acquired biological activities upon oligomerization, we can conclude that surface histidines topography constitutes an exquisite probe for the study of protein structure/function relationship.