Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochloride and a non-ionic denaturant, urea.
The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of alpha-crystallin at room temperature were examined. Native alpha-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 alpha-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of alpha-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 2 M guanidine hydrochloride. alpha B-crystallin is preferentially removed from the native alpha-crystallin aggregate upon treatment with 2 M guanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4 M guanidine 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 alpha-crystallin, is still present when concentrations of alpha-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 2 M guanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting gamma-crystallin from heat-induced aggregation.
Doss-Pepe
E W
EW
Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, USA.
Carew
E L
EL
Koretz
J F
JF
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000378:metabolism*; D007552:Isotonic Solutions; D008854:Microscopy, Electron; D011489:Protein Denaturation / Q000502:physiology; D017433:Protein Structure, Secondary; D014508:Urea / Q000494:pharmacology*