Refolding of RNase A at high concentrations: identification of non-native species

In this paper, we present an analysis of the soluble species formed on refolding of RNase A at various concentrations, in order to characterize these species with respect to structure and activities. Studies were carried out using reverse-phase high-performance liquid chromatography, circular dichroism, chromatography and ultracentrifugation. At all concentrations of protein used, RNase A refolded to the native form, together with formation of non-native species. These non-native species are either misfolded monomers or aggregates; the percentage of such species increases with increasing concentration of enzyme. Such aggregation appears to be a non-random process governed by intermolecular disulfide crosslinking between monomers. These results reaffirm the principle that the information for folding of the protein is encoded in the amino acid sequence itself.