Alkaline unfolding and salt‐induced folding of bovine liver catalase at high pH

Abstract

We have studied the alkaline unfolding of bovine liver catalase and its dependence on ionic strength by enzymic activity measurements and a combination of optical methods like circular dichroism, fluorescence and absorption spectroscopies. Under conditions of high pH (11.5) and low ionic strength, the native tetrameric enzyme dissociates into monomers with complete loss of enzymic activity and a significant loss of A-helical content. Increase in ionic strength by addition of salts like potassium chloride and sodium sulphate resulted in folding of alkaline-unfolded enzyme by association of monomers to tetramer but with significantly different structural properties compared to native enzyme. The salt-induced tetrameric intermediate is characterized by a significant exposure of the buried hydrophobic clusters and significantly reduced A-helical content compared to the native enzyme. The refolding/reconstitution studies showed that the salt-induced partially folded tetrameric intermediate shows significantly higher efficiency of refolding/reconstitution as compared to alkaline-denatured catalase in the absence of salts. These studies suggest that folding of multimeric enzymes proceeds probably through the hydrophobic collapse of partially folded multimeric intermediate with exposed hydrophobic clusters.Keywords : bovine liver catalase; alkaline unfolding; monomer ; salts ; refolding.Catalase is a highly active ubiquitous enzyme which occurs The extent of unfolding of denatured states of proteins under different conditions has long been of interest because of the pos-in almost all aerobically respiring organisms and in part serves sible relevance of their conformations to the protein folding to protect the cells from the toxic effects of hydrogen peroxide. pathways. It has been demonstrated that residual structural pref-Bovine liver catalase, molecular mass 240 kDa, contains four erences, ranging from local clusters of side chains to highly or-identical 57-kDa subunits each equipped with a high-spin Fe(III) dered side chains to highly ordered subdomains, persist in dena-protoporphyrin IX [18]. We have investigated the changes in the tured states of proteins [1Ϫ6]. Hence, there has been a growing structural and functional properties associated with the alkaline recognition of the importance of the compact denatured and par-denaturation of bovine liver catalase and also studied its depentially folded states of proteins, as characterization of these struc-dence on ionic strength. tures and the factors involved in their stability would provide important insight into the interactions responsible for their formation as well as their role in protein folding. EXPERIMENTAL PROCEDURES pH is known to influence the stability of a protein by alteringMaterials. Crystalline bovine liver catalase was prepared acthe net charge on the protein. Many proteins denature at extreme cording to the earlier reported method [19]. The purity of the pH because of the presence of destabilizing repulsive interacprotein was checked by SDS/PAGE foll...

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