Natural and chemically induced oligomeric ribonucleases: structural study by immobilized metal ion affinity electrophoresis and their functional relationship

Abstract

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.

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

“…Dentre as várias técnicas utilizando quelatos metálicos imobilizados, pode-se citar, como exemplos de separação de células, proteínas e ácidos nucléicos, a partição em duas fases aquosas por afinidade, 96,97 a precipitação por afinidade 62,95,98 e a técnica de eletroforese de afinidade. 99,100 …”

Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

“…The Zn(II) binding region in growth hormone was studied using IMAGE by Anisimove et al [66]. Using IMAGE structural study of natural and chemically induced oligomeric ribonucleases was done to study the relationship between surface histidine topography in oligomeric forms of these ribonucleases and their catalytic properties [67].…”

Phosphoprotein electrophoresis in the presence of Fe(III) ions

“…Affinity electrophoresis in combination with following MS analysis represents a valuable tool for phosphoprotein identification. Three different types of immobilized ligands for the preparation of affinity gels have been used: (i) analogs of ligands used in IMAC separation (high relative molecular mass derivatives of iminodiacetic acid (IDA) metal chelate) 9–14, (ii) dinuclear bivalent metal ion complexes (i.e. 1,3‐bis[bis(pyridin‐2‐ylmethyl)amino]propan‐2‐olato dizinc(II) complex) – Phos‐Tag binuclear complex of Me(II) 15, 16 or (iii) immobilized metal ions (in the form of inorganic salts) in a narrow upper part of the separating gel (immobilized metal affinity electrophoresis (IMAEP)) 17–19.…”