Studies of the Denaturation Patterns of Bovine Alpha-Crystallin Using an Ionic Denaturant, Guanidine Hydrochloride and a Non-Ionic Denaturant, Urea

Abstract

The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of α-crystallin at room temperature were examined. Native α-crystallin, at concentrations above and below the previously reported critical micelle concentration (CMC) range, was denatured by varying concentrations of urea and guanidine hydrochloride. The resulting denatured samples were examined by gel filtration fast performance liquid chromatography (FPLC), circular dichroism spectropolarimetry (CD), and transmission electron microscopy. Elution peak samples from gel filtration chromatography with sufficiently high concentrations were examined for subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The studies presented herein demonstrate that the denaturation and renaturation of α-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of α-crystallin which is denatured and the concentration of urea, including certain species which, by gel filtration FPLC, have an apparent molecular weight greater than the native 800 kD aggregate. Transmission electron microscopy has also demonstrated the existence of a high molecular weight aggregate form for denatured samples.Ionic dissociation, in contrast, proceeds much in the same manner above and below the CMC range, the major difference occurring at 2mguanidine hydrochloride. αB-crystallin is preferentially removed from the native α-crystallin aggregate upon treatment with 2mguanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4mguanidine hydrochloride, as indicated by the presence of two apparent homotetrameric species and no further dissociation of these species with increasing guanidine hydrochloride concentrations. CD demonstrates that some secondary structure, which is lost with lower concentrations of α-crystallin, is still present when concentrations of α-crystallin, well above the critical micelle concentration range, are treated with high concentrations of urea at room temperature. In contrast, concentrations both above and below the CMC range demonstrate a significant loss of secondary structure upon treatment with 2mguanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting γ-crystallin from heat-induced aggregation.